Author + information
- Received September 6, 2001
- Revision received March 21, 2002
- Accepted April 18, 2002
- Published online July 17, 2002.
- Harri J Ranki, PhD*,
- Grant R Budas, BSc*,
- Russell M Crawford, PhD*,
- Anthony M Davies, BSc* and
- Aleksandar Jovanović, MD, PhD*,* ()
- ↵*Reprint requests and correspondence:
Dr. Aleksandar Jovanović, Tayside Institute of Child Health, Ninewells Hospital & Medical School, University of Dundee, Dundee, DD1 9SY Scotland, United Kingdom.
Objectives The main objective of the present study was to establish whether 17β-estradiol (E2) regulates expression of cardiac adenosine triphosphate-sensitive potassium (KATP) channel.
Background Based on our previous studies that demonstrate gender-specific differences in sarcolemmal KATPchannels, we have hypothesized that the main estrogen, E2, may regulate expression of cardiac KATPchannels.
Methods Reverse transcription-polymerase chain reaction (RT-PCR) using primers specific for Kir6.2 and sulfonylurea receptor 2A (SUR2A) subunits was performed on total ribonucleic acid (RNA) from rat embryonic heart-derived H9c2 cells. Immunoprecipitation and Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was done on membrane fraction of H9c2 cells. Whole cell electrophysiology and digital epifluorescent Ca2+imaging were performed on living H9c2 cells. All experiments were done in cells incubated 24 h with or without 100 nM E2.
Results The RT-PCR revealed higher levels of SUR2A, but not Kir6.2, messenger RNA (mRNA) in E2-treated, relative to untreated, cells. Increase of the level of only the SUR2A subunit could change the number of sarcolemmal KATPchannels only if the Kir6.2 is in excess over SUR2A. Indeed, RT-PCR analysis demonstrated considerably lower levels of SUR2A mRNA compared with Kir6.2 mRNA. Significantly higher levels of both Kir6.2 and SUR2A protein subunits were found in the membrane fraction of E2-treated cells compared with untreated ones, and the density of current evoked by pinacidil (100 μM), a KATPchannel opener, was significantly higher in E2-treated compared with untreated cells. To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca2+in H9c2 cells exposed to hypoxia-reoxygenation. Intracellular Ca2+loading induced by hypoxia-reoxygenation was significantly decreased by treatment with E2. This E2-mediated protection was inhibited by HMR 1098 (30 μM), but not by 5-hydroxydecanoate (50 μM).
Conclusions In conclusion, this study has demonstrated that E2 increases levels of SUR2A subunit, stimulates KATPchannel formation and protects cardiac cells from hypoxiareoxygenation.
While the risk of heart disease in men increases constantly with age, premenopausal women have a significantly lower risk, which, however, increases rapidly after menopause to levels comparable to male counterparts (1). Estrogen substitution in postmenopausal women reduces cardiovascular mortality by 30% to 50%, suggesting that estrogens are protective (2). In this respect, the antiatherogenic action of estrogens on the lipid profile and arterial wall has been well-documented (3). However, more recent studies suggested that estrogens could directly target cardiomyocytes and protect them against metabolic stress. Specifically, it has been shown that physiologic concentration of 17β-estradiol (E2) protects ventricular cardiomyocytes against hypoxia-reoxygenation (4). The mechanism of this protection is yet unknown. It has been recently suggested that a gender-specific difference in cardiac resistance to metabolic stress might be associated with higher levels of sarcolemmal adenosine triphosphate-sensitive potassium (KATP) channels in females compared with males (5).
It is believed that the activation of a KATPchannel is an important part of endogenous cardioprotective signaling that promotes cellular survival under metabolic challenge (6). In numerous studies, it has been demonstrated that potassium channel openers, drugs that promote opening of KATPchannels, decrease infarct size, mimic ischemic preconditioning and improve functional and energetic recovery of cardiac muscle after ischemic and hypoxic insults (7). More recently, evidence has been provided to suggest that activation of both sarcolemmal and putative mitochondrial KATPchannels may promote cellular survival (7,8). The structure of mitochondrial KATPchannels is still unknown, but the proteins constituting the sarcolemmal KATPchannel complex have been cloned (9–12). Sarcolemmal KATPchannels are heteromultimers composed of, at least, two structurally distinct subunits. In heart, the pore-forming inwardly rectifying K+channel core, Kir6.2, is primarily responsible for K+permeance, whereas the regulatory subunit, also known as the sulfonylurea receptor, or SUR2A, has been implicated in ligand-dependent channel gating (11).
In the present study we have hypothesized that estrogens may regulate expression of sarcolemmal KATPchannels. This hypothesis was based on our previous studies that demonstrated: 1) more sarcolemmal KATPchannels in females than in males (5), and 2) aging-induced decrease in number of sarcolemmal KATPchannels in females (13). We have tested the hypothesis that estrogens may be a regulator of KATPchannel expression in the heart using heart-derived H9c2 cell line. These cells were derived from embryonic rat hearts, and they have properties similar to neonatal and adult cardiomyocytes (14–16). Thus, we have employed reverse transcription-polymerase chain reaction (RT-PCR), Western blotting analysis, patch clamp electrophysiology and digital epifluorescent imaging to test the hypothesis that E2 regulates expression of cardiac KATPchannels.
We report that E2 regulates expression of the SUR2A subunit which, in turn, increases the number of sarcolemmal KATPchannels and promotes cellular resistance to hypoxia-reoxygenation.
Heart H9c2 cells
Rat embryonic heart H9c2 cells (ECACC, Salisbury, United Kingdom) were cultured in a tissue flask (at 5% Co2) containing Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum and 2 mM glutamine. For electrophysiologic and imaging experiments, cells were plated on a 35·10 mm or 60·15 mm culture dish, containing 12-mm or 25-mm glass coverslips. Cells were incubated for 24 h before experimentation without or with 100 nM E2 (Sigma, Dorset, United Kingdom).
Measurement of ribonucleic acid (RNA) levels
Total RNA was then isolated using a commercial kit (RNeasy, Mini Kit, Qiagen, Hilden, Germany) according to the manufacturers instructions. First strand complementary deoxyribonucleic acid (cDNA) was synthesized with random hexanucleotides from 1 mg of total RNA using Reverse Transcription System kit (Promega, Southampton, United Kingdom). Polymerase chain reactions (PCRs) were done using ReadyMix Red Tag from Sigma in a thermal cycler Model Phoenix (Helena Biosciences, Sunderland, United Kingdom) under the following conditions: for Kir 6.2: 94°C for 3 min, 34 cycles (94°C for 0.5 min, 66.1°C for 0.5 min, 70°C for 1 min), final extension at 70°C for 5 min; for SUR2A the conditions were the same as for Kir6.2 except that the number of cycles was 49 and the annealing temperature was 66.1°C. The primers had the following sequences, for the 387 base-long product for rat Kir6.2: sense, 5′-ATGCGCAAGACCACCAGC-3′; antisense, 5′-TGGCGGGCTGTGCAGAG-3′. For the 375 base-long product for rat SUR2A: sense, 5′-CTAGACGCCACTGTCAC-3′; antisense, 5′-AGAGAACGAGACACTTGG-3′. In experiments set to determine the concentration-dependent effect of E2 on SUR2A messenger RNA (mRNA), the following primers were used: sense, 5′-CTAGACGCCACTGTCAC-3′; antisense, 5′-AGAGAACGAGACACTTGG-3′. Reverse transcription polymerase chain reaction conditions were as described above for SUR2A mRNA, and 36 to 40 cycles were used. The loading of RNA was checked by human glyderaldehyde-3-phosphate dehydrogenase (GAPDH)-primers: sense, 5′-CATCACCATCTTCCAGGAGCGA-3′; antisense, 5′-GTCTTCTGGGTGGCAGTGATGG-3′; the size of GAPDH-product was 341 base pairs). There were no significant differences in intensity of GAPDH-levels between experimental groups. The nature of PCR product was confirmed by deoxyribonucleic acid sequencing. The PCR product band intensities were analyzed using the Quantiscan software.
Immunoprecipitation and Western blotting analysis
Sheep antipeptide antibodies were raised against synthetic peptides comprised of residues 33 to 47 in the Kir6.2 protein (ARFVSKKGNCNVAHK) and residues 311 to 32 in the SUR2A protein (CIVQRVNETQNGTNN), conjugated to a carrier protein, keyhole limpet hemocyanin, and used for immunoprecipitation and Western blotting. To obtain the membrane fraction, H9c2 cardiac cells were homogenized in buffer I (tris[hydroxymethyl]-aminomethane 10 mM, NaH2Po420 mM, ethylenediaminetetraacetic acid 1 mM, phenylmethyl sulfonyl fluoride 0.1 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml, at pH = 7.8) and incubated for 20 min (at 4°C). The osmolarity was restored with KCl, NaCl and sucrose, and the obtained mixture was centrifugated at 500 g. The supernatant was diluted in buffer II (imidazole 30 mM, KCl 120 mM, NaCl 30 mM, NaH2Po420 mM, sucrose 250 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml, at pH = 6.8) and centrifugated at 7,000 g, pellet removed and supernatant centrifugated at 30,000 g. The obtained pellet contains membrane fraction. Ventricular tissue was snap-frozen immediately upon extraction and ground to a powder under liquid nitrogen. The powder was resuspended in 10 ml of tissue buffer (20 mM Hepes, 150 mM NaCl, 1% Triton-X 100, pH 7.5) and homogenized. Protein concentration was determined using the method of Bradford; 10 μg of the epitope-specific Kir6.2 antibody or 40 μg of the epitope-specific SUR2A antibody was prebound to Protein-G Sepharose beads and used to immunoprecipitate from 50 μg of membrane fraction protein extract. The pellets of this precipitation were run on SDS polyacrylamide gels, for Western analysis. Western blot probing was performed using 1/200 and 1/300 dilutions of anti-SUR2A and anti-Kir6.2 antibody, respectively, and detection was achieved using Protein-G horse radish peroxydase and enhanced chemiluminescence reagents.
Cells were superfused with Tyrode solution (in mM: 136.5 NaCl, 5.4 KCl, 1.8 CaCl2, 0.53 MgCl2, 5.5 glucose, 5.5 HEPES-NaOH, pH 7.4). Pipettes (resistance 3 to 5 MΩ) were filled with (in mM): KCl 140, MgCl21, ATP 3, HEPES-KOH 5 (pH 7.3). Recordings were made at room temperature (22°C). During each experiment, the membrane potential was normally held at −40 mV, and the currents evoked by a series of 400 ms current steps (+40 mV to +80 mV in 20 mV steps) recorded directly to hard disk using an Axopatch-200B amplifier, Digidata-1321 interface and pClamp8 software (Axon Instruments, Inc., Forster City, California). The capacitance compensation was adjusted to null the additional whole-cell capacitative current. The slow capacitance component measured by this procedure was used as an approximation of the cell surface area and allowed normalization of current amplitude (i.e., current density). Currents were low-pass filtered at 2 kHz and digitized.
Digital epifluorescent microscopy
H9c2 cells were superfused with Tyrode solution and loaded with the esterified form of the Ca2+-sensitive fluorescent probe Fura-2 (Fura-2AM, dissolved in dimethyl sulfoxide plus pluronic acid; Molecular Probes, Eugene, Oregon). Cells were imaged using a digital epifluorescence imaging system coupled to an inverted microscope (Image Solutions, Standish, United Kingdom). A mercury lamp served as a source of light to excite Fura-2AM at 340 nm and 380 nm. Fluorescence emitted at 520 nm was captured, after crossing dichroic mirrors, by an intensified charge-coupled device camera and digitized using an imaging software. An estimate of the cytosolic Ca2+concentration, as a function of Fura-2 fluorescence, was calculated according to the equation: where R is the fluorescence ratio recorded from the cell, Rminand Rmaxis the minimal and maximal fluorescence ratio, Kdis the dissociation constant of the dye (236 nM) and β is the ratio of minimum to maximum fluorescence at 380 nm. Hypoxia-reoxygenation was induced as follows: single field-stimulated (30 mV, 5 ms, 0.5 Hz) cells were perfused with Tyrode solution containing (in mM) NaCl 136.5, KCl 5.4, CaCl21.8, MgCl20.53, glucose 5.5, HEPES-NaOH 5.5 (pH 7.4) at a rate of 1 ml/min. Under these conditions the Po2in perfusate was approximately 140 mm Hg. For hypoxia the solution was continuously bubbled with 100% argon while the exchange of o2between solution in the chamber and air was prevented by nitrogen jet. The Po2under these conditions was approximately 20 mm Hg. The duration of hypoxia was 10 min, followed by reoxygenation with Tyrode solution for 10 min. Some experiments were done under the presence of 30 μM HMR 1098 (Aventis Pharma, Frankfurt, Germany) or 50 μM 5-hydroxydecanoate (5-HD, RBI, Natick, Massachusetts).
Data are presented as mean ± SEM, with nrepresenting the number of experiments. Mean values obtained were compared by the paired or unpaired Student’s ttest where appropriate. Results for Kir6.2 and SUR2A obtained with RT-PCR for each sample were normalized taking into account GAPDH levels regardless that they were not significantly different in tested samples. The difference between means were assessed using ttest (paired or unpaired), by Tukey test, chi-square test or by the two-way repeated measures analysis of variance using SigmaStat program (Jandel Scientific, Chicago, Illinois). A p value of < 0.05 was considered statistically significant.
Higher levels of SUR2A mRNA, but not Kir6.2 mRNA, in E2-treated cells
Reverse transcription polymerase chain reaction analysis of H9c2 cells demonstrated higher levels of SUR2A mRNA in E2-treated cells relative to untreated ones (PCR product band intensity was 7.7 ± 0.3 arbitrary units [AU] for untreated and 13.5 ± 0.5 for E2-treated cells, n = 3 of each, p = 0.01; Fig. 1B). In contrast, Kir6.2 mRNA levels were similar between two groups of cells, irrespective whether they were treated with E2 (Fig. 1A; intensity of bands were 28.0 ± 2.0 AU in control and 25.9 ± 2.1 AU in E2-treated cells, n = 3 for each, p = 0.54). No differences were observed between GAPDH levels after treatment with E2 (Fig. 1C; intensity of bands were 6.5 ± 0.5 AU in control and 6.0 ± 1.0 AU in E2-treated cells, n = 3 for each, p = 0.69). The effect of E2 on SUR2A mRNA levels was concentration-dependent (Fig. 1D; n = 3 for each, p = <0.001).
Kir6.2 mRNA is present in higher amounts in H9c2 cells than SUR2A mRNA
From the same cDNA pool, different volumes of Kir6.2 and SUR2A cDNA were taken and subjected to PCR using the best conditions for both reactions. The first product band for Kir6.2 was visible with 0.01 μl of cDNA at 34 cycles of the PCR reaction, while SUR2A product was visualized with 1 μl of cDNA at 49 cycles. Because each cycle of the PCR reaction doubles amount of products, there were considerably smaller amounts of SUR2A mRNA compared with Kir6.2 mRNA in H9c2 cells.
E2 increases the number of KATPchannels in H9c2 cells
To determine whether E2 regulates the number of Kir6.2/SUR2A channels in plasmalemma, we have immunoprecipitated the membrane fraction with anti-Kir6.2 antibody and “probed” the precipitate with the anti-SUR2A antibody and vice verse. Western blotting analysis revealed higher levels of both Kir6.2 and SUR2A proteins in E2-treated cells compared with untreated ones (Kir6.2: the band intensity was 10.8 ± 2.7 AU for untreated and 22.5 ± 2.5 AU for E2-treated cells, n = 4 of each, p = 0.01; Fig. 2A; SUR2A: the band intensity was 11.8 ± 1.3 AU for untreated and 26.0 ± 3.2 AU for E2-treated cells, n = 4 of each, p = 0.006; Fig. 2B). In both untreated and E2-treated cells, pinacidil (100 μM), a opener of KATPchannels, induced the outward membrane current. The pinacidil-sensitive component was significantly larger in E2-treated than untreated cells (current density at 80 mV was 2.1 ± 0.2 pA/pF in control and 3.2 ± 0.3 pA/pF in E2-treated cells, p = 0.01, n = 7 for each).
E2-treated cells are more resistant to hypoxia-reoxygenation injury then untreated cells
To test the effect of E2 on cellular response to hypoxia-reoxygenation, we have measured on-line, intracellular concentration of Ca2+in H9c2 cells exposed to hypoxia-reoxygenation. Figure 3Adepicts the typical experiment. At rest, untreated and E2-treated cells had similar levels of cytosolic Ca2+(untreated: 56 ± 5 nM, n = 7; E2-treated: 50 ± 5 nM, p = 0.46, n = 9; Fig. 3A). In untreated cells, hypoxia-reoxygenation induced significant intracellular Ca2+loading (154 ± 7 nM, p = 0.001 when compared with the control; n = 7, Fig. 3A). In contrast, cells treated with E2 had a significantly lower increase of intracellular Ca2+during hypoxia-reoxygenation than untreated ones (E2-treated: 82 ± 6 nM, n = 9, p = 0.004 when compared with untreated cells, Fig. 3A). There was a statistically significant interaction between E2-treatment and cellular response to the hypoxia-reoxygenation (p = 0.011). In untreated group of cells, 22% did not respond with Ca2+loading to hypoxia-reoxygenation challenge, while, in the E2-treated cell group, this number was significantly higher, 68% (Fig. 3B, p = < 0.001); HMR 1098 (30 μM), a sarcolemmal KATPchannel blocker, partially restored sensitivity to hypoxia/reoxygenation in the E2-treated group (Fig. 3B, 41% of cells responded with Ca2+loading, which was not significantly different compared with untreated cells, p = 0.134). In contrast, 5-HD (50 μM), an antagonist of mitochondrial KATPchannels, did not inhibit protection afforded by E2 treatment (Fig. 3B, p < 0.001 when compared with the control).
This study demonstrates that E2 stimulates the expression of SUR2 subunit in the heart-derived cell line, leading to an increase in the number of sarcolemmal KATPchannels and increased resistance to hypoxia-reoxygenation. This is the first report of a hormone-regulating expression of cardiac KATPchannels, ion channels that transduce metabolic status of a cell into membrane excitability.
The effect of E2 on Kir6.2 and SUR2A mRNA levels
In this study semiquantitative RT-PCR revealed that treatment with E2 increases the levels of SUR2A mRNA, but not Kir6.2 mRNA. The higher levels of SUR2A mRNA, but not Kir6.2 mRNA, in E2-treated cells compared with untreated ones resembles our results previously obtained on female and male adult cardiomyocytes, that is, higher levels of SUR2A mRNA in female compared with male cardiomyocytes (5). The concentration-dependent nature of E2 action shows that this effect is genuine and mediated through a saturable binding site, which is in accord with the receptor-mediated effect of a hormone. Considering that Kir6.2 and SUR2A subunits form the KATPchannel in 1:1 ratio (17), higher levels of only one subunit does not necessarily mean that more KATPchannels will be formed. In adult cardiomyocytes, we have previously demonstrated that the Kir6.2 subunit is in excess over the SUR2A subunit (5). The biological consequence of this disproportion between Kir6.2 and SUR2A levels is that the number of sarcolemmal KATPchannels is solely controlled by the levels of SUR2A (5). In this study, similarly as in adult cardiomyocytes (5), RT-PCR revealed higher amounts of Kir6.2 mRNA than SUR2A mRNA, suggesting that fluctuation of SUR2A levels regulates the number of KATPchannels in H9c2 cells and that an increase in SUR2A mRNA may be sufficient to increase the levels of the channel proteins.
The effect of E2 on number of KATPchannels in plasmalemma
In sarcolemma, Kir6.2 and SUR2A physically associate to form KATPchannels (17). To secure measuring of only those subunits forming the channel, we have immunoprecipitated cardiac membrane fraction with anti-Kir6.2 antibody and then “probed” with anti-SUR2A antibody and otherwise around. Using this approach to measure only subunits coprecipitated with each other, we have excluded subunits that are present in the cell but do not form the channel. Both Kir6.2 and SUR2A subunits are found in much higher levels in membrane fraction from E2-treated than untreated cells, thus providing direct evidence that E2 increases density of plasma membrane KATPchannels in H9c2 cells. This conclusion was further supported by the whole cell electrophysiology. Because in intact cardiomyocytes KATPchannels are normally closed, channels were activated by pinacidil, an established opener of KATPchannels (18,19). Our findings that the magnitude of the response to pinacidil followed the pattern of Kir6.2 and SUR2A membrane levels is in accord with the notion that E2 increases cardiac KATPchannel density. The apparent discrepancy between the results obtained with RT-PCR methodology (differences only in SUR2A levels) and Western blotting (differences in both Kir6.2 and SUR2A levels) could be explained by the fact that RT-PCR measured levels of total SUR2A and Kir6.2 mRNA, while Western blot selectively measured levels of those SUR2A and Kir6.2 subunits that are physically associated to form the channels. These findings further support the notion that the SUR2A subunit controls the number of functional channel proteins in cardiac cells and that E2 controls the number of sarcolemmal KATPchannels by regulating expression of the SUR2A subunit.
The effect of E2 on resistance to hypoxia-reoxygenation
The consequence of E2-mediated regulation of KATPchannel expression has yet to be fully understood. It has been previously shown that physiologic replacement of estradiol protects the myocardium after global ischemia in the ovariectomized animals (20). Also, we have demonstrated that cardiomyocytes from females are more resistant to metabolic stress compared with those in males (5)and that E2 directly protects adult cardiomyocytes against chemical hypoxia/reoxygenation (4). It has been also reported that E2 treatment promotes survival of H9c2 cells exposed to glucose-deprived/hypoxic condition (16). In this study we have measured intracellular concentration of Ca2+as a parameter that reflects the metabolic condition of a cardiac cell (4,5,18). As expected, hypoxia-reoxygenation induced intracellular Ca2+loading in H9c2 cells, indicating that these cells are vulnerable to such an insult. The fact that treatment with E2 protected these cells against hypoxia/reoxygenation-induced Ca2+loading are compatible with the notion that E2 possess direct cardioprotective properties (4)associated with the E2 property to regulate expression of KATPchannels. Inhibition of the E2-mediated protection with HMR 1098, a selective sarcolemmal KATPchannel blocker (21), directly associates the beneficial effect of E2 with upregulation of sarcolemmal KATPchannels. The cardioprotective role of sarcolemmal KATPchannels has been contested in the last few years (22), but more recent reports reaffirm the idea that opening of sarcolemmal KATPchannels protects against metabolic stress (23–25). Our finding that the predominant blocker of mitochondrial KATPchannels, 5-HD (22), did not inhibit the protective effect of E2, excludes the possibility that these channels were actively involved in protection afforded by E2. The observed E2-mediated upregulation of sarcolemmal KATPchannels and increased resistance to metabolic stress is in agreement with previous studies showing positive correlation between plasma levels of estrogens and number of sarcolemmal KATPchannels and the cardiomyocyte’s resistance to metabolic insult (5,13). Therefore, it seems that one of the consequences of E2 action on cardiomyocytes, either adult or embryonic, is more KATPchannels expressed on plasmalemma and increased resistance towards metabolic stress.
The main limitation of the study might be that it was done on embryonic heart-derived cell line and not on adult cardiomyocytes. However, bearing in mind that properties of H9c2 cells are similar to those in adult cardiomyocytes (13–15), it is likely that the findings from this study are of relevance for adult cardiomyocytes as well.
In conclusion, this study has demonstrated that E2 increases levels of the SUR2A subunit, stimulates KATPchannel formation and protects cardiac cells from hypoxia-reoxygenation. The obtained results could provide a basis for developing therapeutic strategies against ischemic heart disease centered around KATPchannels.
The authors thank Avis Pharma (Frankfurt, Germany) for providing HMR 1098.
☆ Supported by grants from the American Heart Association, Anonymous Trust, Biotechnology and Biological Sciences Research Council, British Heart Foundation, National Heart Research Fund, TENOVUS-Scotland and the Wellcome Trust to Dr. Jovanović.
- arbitrary units
- complementary deoxyribonucleic acid
- glyceraldehyde-3-phosphate dehydrogenase
- adenosine triphosphate-sensitive potassium channel
- messenger ribonucleic acid
- polymerase chain reaction
- ribonucleic acid
- reverse transcription-polymerase chain reaction
- sulfonylurea receptor 2A
- Received September 6, 2001.
- Revision received March 21, 2002.
- Accepted April 18, 2002.
- American College of Cardiology Foundation
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