Author + information
- Received December 15, 2000
- Revision received May 16, 2001
- Accepted May 22, 2001
- Published online September 1, 2001.
- Harri J Ranki, PhD∗,
- Grant R Budas, BSc∗,
- Russell M Crawford, PhD∗ 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
The main objective of this study was to establish whether gender regulates expression and/or properties of cardiac ATP-sensitive K+(KATP) channels.
Recently, evidence has been provided that differing cardiac responses in males and females to metabolic stress may result from gender-specific difference(s) in the efficiency of endogenous cardioprotective mechanism(s) such as KATPchannels.
A reverse transcription polymerase chain reaction (RT-PCR) using primers specific for Kir6.2, Kir6.1 and SUR2A subunits was performed on total RNA from guinea pig ventricular tissue. Western blotting using anti-Kir6.2 and anti-SUR2A antibodies was performed on cardiac membrane fraction. Whole-cell, single-channel electrophysiology and digital epifluorescent Ca2+imaging were performed on isolated guinea pig ventricular cardiomyocytes.
The RT-PCR revealed higher levels of SUR2A, but not Kir6.1 and Kir6.2, messenger RNA in female tissue relative to male tissue, while much higher levels of both Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue compared with male tissue were found. In both male and female tissue, pinacidil (100 μM), a KATPchannel opener, induced outward whole-cell currents. The current density of the pinacidil-sensitive component was significantly higher in female tissue than it was in male tissue, while no differences in single KATPchannel properties between genders were observed. Ischemia-reperfusion challenge induced significant intracellular Ca2+loading in male, but not female, cardiomyocytes. To test the hypothesis that SUR2A expression is the limiting factor in KATPchannel formation, we took different volumes of Kir6.2 and SUR2A complementary DNA (cDNA) from the same cDNA pool and subjected them to PCR. In order to obtain a band having 50% of the maximal intensity, a volume of SUR2a cDNA approximately 20 times the volume of Kir6.2 cDNA was required.
This study has demonstrated that female tissue expresses higher levels of functional cardiac KATPchannels than male tissue due to the higher expression of the SUR2A subunit, which has an impact on cardiac response to ischemia-reperfusion challenge.
Gender-specific differences in the incidence of cardiovascular disease were first recognized more than half a century ago (1). It is now well established that, although the risk of heart disease in men increases constantly with age, premenopausal women have a significantly lower risk that increases rapidly after menopause to levels comparable to that of their male counterparts. In fact, in postmenopausal women receiving estrogen substitution, the cardiovascular mortality is 30% to 50% less than that found in their untreated female counterparts (2). Traditionally, the major mechanism responsible for the protective effect of female gender is believed to be due to an antiatherogenic action of female sex hormones on the lipid profile (3). More recently, in addition to this traditional view, some evidence has been provided that a gender-specific difference in cardiac response to a metabolic stress may also be due to some gender-specific difference(s) in efficiency of endogenous cardioprotective mechanism(s) unrelated to estrogen vascular effects (4).
It is believed that the activation of an ATP-sensitive K+(KATP) channel is an important part of endogenous cardioprotective signaling that promotes cellular survival under conditions of severe metabolic stress (5). This ion channel was originally discovered in membrane patches excised from ventricular cardiomyocytes, and it has been hypothesized that it couples the metabolic state of the cell with membrane excitability (6). In numerous studies, it has been demonstrated that potassium channel openers—drugs that promote the 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 sarcolemmal and 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). The KATPchannels are heteromultimers composed of at least two structurally distinct subunits. In heart tissue, 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). The established knowledge about the structure of cardiac KATPchannels in the last few years permits further investigation of factors involved in the regulation of KATPchannel expression and function. In this regard, we employed reverse transcription polymerase chain reaction (RT-PCR), Western blotting analysis, patch clamp electrophysiology and digital epifluorescent imaging to test the hypothesis that gender influences expression and/or properties of cardiac KATPchannel.
We report that female gender is associated with higher levels of functional cardiac KATPchannels, which is specifically due to the higher levels of the SUR2A subunit. We have also demonstrated that the observed gender-specific difference may have a profound impact on cardiac response to metabolic challenge.
RNA extraction, RT-PCR
Total RNA from cardiac ventricular tissue was isolated using a commercial kit (RNeasy, Midi Kit, Qiagen; Crawley, United Kingdom) according to the manufacturer’s instructions. The first strand of complementary DNA (cDNA) was synthesized from 1 μg tissue RNA using 200 U of MML-V reverse transcriptase (Promega; Southampton, United Kingdom) and a random hexanucleotide mixture (0.5 μg of primer per μg RNA). The polymerase chain reaction (PCR) was performed with gene-specific primers. The total PCR volume was 25 μl, including 1/30 of the RT reaction, 25 pmol of each primer and 2.5 U PromegaTag (Promega). The PCR was performed in a thermal cycler Model Phenix (Helena Biosciences) under the following conditions: the PCR was run with a hot-start for 5 min at 95°C (initial melt), then for 38 (Kir 6.2), 40 (Kir6.1) or 41 (SUR2A) cycles of 0.5 min at 94°C, 0.5 min at 55°C and 1 min at 72°C (final extension). The primers had the following sequences. For gpKir6.1: sense, 5′-GTCCTTCCTCTGCAGTTGGC-3′; antisense, 5′-CATGACGCGTTGATGATCAGACC-3′. For gpKir6.2: sense, 5′-CATTCTGTTCCCACCTTGAGA-3′; antisense, 5′-GGTGCAGACTTTATTGGCAA-3′. For gpSUR2A: sense, 5′-GTTGACATATTTGATGGAAAG-3′; antisense, 5′-CTACTTGTGAGTCATCACCAAGGT-3′. These conditions were set based on our preliminary studies that demonstrated that, under these conditions, intensity of the PCR product band is approximately 50% of its maximum. All RT-PCR experiments were carried out in the presence of appropriate controls and were repeated at least five times, and they regularly resulted in a single product of a size similar to the expected ones (285 base pair [bp] for Kir6.1, 349 bp for Kir6.2 and 563 bp for SUR2A). GAPDH-specific primers were used to test RNA loading (gpGAPDH: sense, 5′-CATCACCATCTTCCAGGAGCGA-3′; antisense, 5′-GTCTTCTGGGTGGCAGTGATGG-3′, 341 bp was size of product). There were no significant differences in intensity of GAPDH-specific product band between experimental groups. The PCR product-band intensity was 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 321 to 334 in the SUR2A protein (CIVQRVNETQNGTNN), conjugated to a carrier protein, keyhole limpet hemocyanin and used for immunoprecipitation and Western blotting. To obtain the membrane cardiac fraction, guinea pig ventricular tissue was homogenized in buffer I (TRIS 10 mM, NaH2PO420 mM, EDTA 1 mM, PMSF 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; the pellet was removed, and the supernatant centrifugated at 30,000 g. The obtained pellet contained 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 HRP and ECL reagents.
Isolation of single cardiomyocytes
Ventricular cardiomyocytes were dissociated from sexually mature male and female guinea pigs (200 to 250 g) using an established enzymatic procedure (13). In brief, hearts were retrogradely perfused (at 37°C) with medium 199 for 2 to 3 min, followed by Ca2+-EGTA-buffered low-Ca2+medium (pCa = 7) for 80 s and, finally, low-Ca2+medium-containing pronase E (8 mg per 100 ml), proteinase K (1.7 mg per 100 ml), bovine albumin (0.1 g per 100 ml, fraction V) and 200 μM CaCl2. Ventricles were cut into small fragments (6 mm to 10 mm3) in the low-Ca2+medium enriched with 200 μM CaCl2. Single cells were isolated by stirring the tissue (at 37°C) in a solution containing pronase E and proteinase K supplemented with collagenase (5 mg per 10 ml). After 10 min, the first aliquot was removed, filtered through a nylon sieve, centrifuged for 60 s (at 300 to 400 rpm) and washed twice. Remaining tissue fragments were re-exposed to collagenase, and isolation was continued for two to three such cycles. Isolated cardiomyocytes were stored in low-Ca2+medium with 200 μM CaCl2. Only rod-shaped cardiomyocytes with clear striations and a smooth surface were used for electrophysiological recordings.
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). Fire-polished 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 were evoked by a series of 400 ms depolarizing and hyperpolarizing current steps (−80 to +80 mV in 20 mV steps) recorded directly to hard disk using an Axopatch-200B (Axon Instruments, Inc., Foster City, California) amplifier, Digidata-1321 interface and pClamp8 software. 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). The average cell capacitance was 123 ± 12 pF and 133 ± 12 pF for cardiomyocytes isolated from male and female animals, respectively (n = 8 for each). Currents were low-pass filtered at 10 kHz digitized at 2 kHz. Pooled data are presented as mean ± SEM, and values of nrefer to the number of experiments in each group.
Single-channel recordings and kinetic analysis
To monitor on-line behavior of single-channel molecules, the giagaohm seal patch-clamp technique was applied in the inside-out configuration, as we previously described (14). Cells were superfused with (in mM): KCl 140, MgCl21, EGTA 5, HEPES-KOH 5 (pH 7.4). Fire-polished pipettes, coated with Sylgard (resistance 5 to 7 MΩ) were filled with (in mM): KCl 140, CaCl21, MgCl21 and HEPES-KOH 5 (pH 7.3). Recordings were made at room temperature (22°C) using a patch-clamp amplifier (Axopatch-200B). Single-channel activity was monitored on-line and stored on a PC. Data were reproduced, low-pass filtered at 1 KHz (−3 dB), sampled at 80 μs rate and further analyzed using the “pClamp8” software. The threshold for judging the open state was set at half of the single-channel amplitude. For analysis of channel behavior, amplitude and open- and closed-dwell time histograms were constructed and fitted by a single exponent.
Digital epifluorescent microscopy
Relaxed, rod-shaped, cardiomyocytes 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 with 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 imaging software. An estimate of the cytosolic Ca2+concentration, as a function of Fura-2 fluorescence, was calculated according to the equation: [Ca2+] = (R − Rmin/Rmax− R)Kdβ; where R is the fluorescence ratio recorded from the cell, Rminand Rmaxthe minimal and maximal fluorescence ratio, Kdthe dissociation constant of the dye (236 nM) and β the ratio of minimum to maximum fluorescence at 380 nm (15,16). The ischemia-reperfusion challenge was induced as follows: single cardiomyocytes 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. After an initial period of equilibration, the perfusate was changed to an ischemic solution. To mimic the in vivo condition, the ischemic solution, otherwise similar to Tyrode solution, contained slightly increased K+concentration (16 mM); pH was reduced (pH = 6.5), and glucose was omitted (17). The solution was also continuously bubbled with 100% argon, and the exchange of O2between solution in the chamber and air was prevented by nitrogen jet (18). The PO2under these conditions was approximately 20 mm Hg. The duration of ischemic perfusion was 20 min, followed by reperfusion with Tyrode solution for 10 min.
Data are presented as mean ± SEM, with nrepresenting the number of patched cells or examined hearts. Mean values obtained were compared by the paired or unpaired Student ttest where appropriate. Results for Kir6.2, Kir6.1 and SUR2A obtained with RT-PCR for each sample were normalized, taking into account GAPDH levels even though they were not significantly different in tested samples. The effect of gender on pinacidil-mediated protection of cardiomyocytes was assessed by the two-way repeated measures analysis of variance using the SigmaStat program (Jandel Scientific, Chicago, Illinois). A p value <0.05 was considered statistically significant.
Higher levels of SUR2A messenger RNA (mRNA), but not Kir6.1 and Kir6.2 mRNA, in female relative to male hearts
An RT-PCR analysis of ventricular cardiac tissue demonstrated higher levels of SUR2A mRNA in female tissue relative to male tissue (PCR product-band intensity was 19.7 ± 1.5 arbitrary units [AU] for male tissue and 35.7 ± 5.6 AU for female tissue, n = 3 of each, p = 0.03; Fig. 1B). In contrast, although Kir6.1 and Kir6.2 mRNA levels were higher in male tissue than they were in female tissue, the differences were not statistically significant (Fig. 1, A to C). Intensity of bands was estimated to be 22.0 ± 0.0 AU in male tissue and 17.2 ± 1.3 AU in female tissue (n = 2 to 3; p = 0.08; Fig. 1A) and 17.0 ± 2.1 AU in male tissue and 12.8 ± 1.3 AU in female tissue (n = 3; p = 0.12; Fig. 1C) for Kir6.1 and Kir6.2, respectively.
More Kir6.2 and SUR2A proteins in cardiac membrane fraction in female tissue than in male tissue
Western blotting with the specific anti-Kir6.2 and anti-SUR2A antibodies uncovered signals at sizes expected for Kir6.2 and SUR2A, that is, approximately 35 kDa and approximately 150 kDa, respectively (Fig. 2A). The intensity of both signals was much higher in the cardiac membrane fraction from female than from male tissue (Fig. 2B).
Whole-cell membrane current response to pinacidil, a KATPchannel opener, is more pronounced in female than in male cardiomyocytes
Figure 3depicts the effect of pinacidil (100 μM) on outward current in cardiomyocytes from male and female hearts. From the holding potential of −40 mV, 400 ms voltage pulses between −80 to +80 mV were applied to the cell in a 20-mV increment every 1 s. In the control conditions, outward current was observed in response to both hyperpolarizing and depolarizing voltage steps. Under control conditions, there was no statistically significant difference in current density between male and female myocytes in the absence of pinacidil (male tissue: 11.6 ± 1.2 pA/pF; female tissue: 14.1 ± 1.4 pA/pF, n = 7 for each, p = 0.14; Fig. 3A to C). In both male and female tissue, pinacidil (100 μM) induced time-independent outward currents (Fig. 3A). In the presence of pinacidil, current density rose from 11.6 ± 1.2 pA/pF to 13.6 ± 1.4 pA/pF in male tissue (p = 0.002, n = 7; Fig. 3C) and from 14.1 ± 1.4 pA/pF to 19.0 ± 1.9 pA/pF in female tissue (p = 0.003, n = 7; Fig. 3C). The average current density of the pinacidil-sensitive component was 2.0 ± 0.3 pA/pF in male tissue and 4.9 ± 0.5 pA/pF in female tissue (n = 7 for each; Fig. 3D). This difference was statistically significant (p = 0.02; Fig. 3E).
To test whether basic single-channel properties differ according to gender, we employed single-channel patch clamp electrophysiology. After excision of a membrane patch from a cardiomyocyte in an ATP-free environment, vigorous opening of KATPchannels occurred. Typical segments of single KATPchannel recordings are depicted on Figure 4A. There were no statistically significant differences in single-channel amplitude (male tissue: 4.14 ± 0.08 pA; female tissue: 4.29 ± 0.20 pA at HP = −60 mV; n = 3, p = 0.35; Fig. 4B), mean open time (male tissue: 1.72 ± 0.11 ms; female tissue: 1.98 ± 0.12 ms at HP = −60 mV; n = 3, p = 0.19; Fig. 4C) and mean closed time (male tissue: 0.28 ± 0.03 ms; female tissue: 0.23 ± 0.02 ms at HP = −60 mV; n = 3, p = 0.27; Fig. 4D) between cells harvested from male or female guinea pigs.
Cardiomyocytes from female cells are more resistant to ischemia-reperfusion injury compared with male cells
Intracellular concentration of Ca2+is an accurate parameter of a cardiomyocyte metabolic condition (19). Therefore, to test the gender-specific difference in cellular response to ischemia-reperfusion challenge, we measured on-line, intracellular concentration of Ca2+in ventricular cells exposed to ischemia-reperfusion. At rest, cardiomyocytes isolated from male animals had similar levels of cytosolic Ca2+(male cells: 40 ± 3 nM, n = 8; female cells: 54 ± 10 nM, p = 0.26, n = 10; Fig. 5). In male cells, but not female cells, ischemia-reperfusion induced significant intracellular Ca2+loading (male cells: 173 ± 50 nM, p = 0.02 when compared with the control; n = 8; female cells: 62 ± 10 nM, p = 0.59 when compared with the control, Fig. 5). There was a statistically significant relationship between gender and cellular response to the ischemia reperfusion (p = 0.013).
Kir6.2 mRNA is present in higher amounts in ventricular tissue than SUR2A mRNA
Bearing in mind that Kir6.2 and SUR2A form the channel in a ratio of 1:1 (20), higher SUR2A mRNA levels may lead to higher KATPchannel density only if the number of SUR2A subunits is a rate-limiting step. To be a rate-limiting factor in forming the KATPchannel, SUR2A needs to be available in lower amounts than Kir6.2. Therefore, to test this hypothesis, we performed RT-PCR analysis to compare Kir6.2 and SUR2A mRNA levels. Specifically, from the same cDNA pool, different volumes of Kir6.2 and SUR2A cDNA were taken and subjected to PCR using the same conditions. As depicted in Figure 6A, the first product band for Kir6.2 was visible with 0.01 μl of cDNA, while for SUR2A this was the case with 0.3 μl of cDNA. To obtain a band having 50% of the maximal intensity, approximately 0.05 μl of cDNA for Kir6.2 and approximately 1.5 μl of cDNA for SUR2A was required (Fig. 6B).
This study demonstrates that female gender is associated with higher levels of functional sarcolemmal cardiac KATPchannels, which is specifically due to the higher levels of the SUR2A subunit. This is the first report of a gender-specific difference in cardiac KATPchannels, an ion channel that transduces the metabolic status of a cell into membrane excitability.
In this study, RT-PCR analysis demonstrated higher levels of SUR2A mRNA, but not Kir6.2 mRNA and Kir6.1 mRNA, in female compared with male hearts. Although it is generally accepted that Kir6.2 and SUR2A reconstitute this cardiac KATPchannel (11), we tested Kir6.1 mRNA as well, bearing in mind previous studies in which changes in Kir6.1 mRNA, but not Kir6.2 mRNA levels, were associated with the changes in SUR2A mRNA levels (21). In this study, this was not the case, and the only observed difference was at the SUR2A mRNA levels. Considering that Kir6.2 and SUR2A subunits form the KATPchannel in a 1:1 ratio (19), the presence of higher levels of only one subunit does not necessarily mean that more KATPchannels will be formed.
Therefore, to test the hypothesis that more KATPchannels are present in the sarcolemma of female cells than male cells, we measured levels of KATPchannel protein subunits in cardiac membrane fraction using Western blotting analysis. For this purpose we applied antibodies raised against epitopes on KATPchannel subunits, Kir6.2 and SUR2A. Anti-Kir6.2 antibody was previously established as a specific (22), while for this study we created an anti-SUR2A antibody that has been thoroughly characterized and determined to be highly specific (Fig. 2). The Western blotting analysis of the cardiac membrane fraction demonstrated that both Kir6.2 and SUR2A subunits are present in much higher levels in cardiac membrane fraction from female tissue than from male tissue, thus providing direct evidence that density of sarcolemmal KATPchannels differs between genders. Furthermore, our finding that the current density of membrane current evoked by pinacidil, a KATPchannel opener, was significantly higher in female tissue than it was in male tissue is in agreement with the idea that there are more functional cardiac KATPchannels in female tissue than in male tissue. The fact that that single KATPchannel properties did not differ significantly between genders further confirms that the higher current density in response to pinacidil in female tissue is due to a higher number of KATPchannels on the sarcolemma and not due to any gender-specific difference(s) at the level of single-channel properties. The apparent discrepancy between the results obtained with an RT-PCR methodology (differences only in SUR2A level) on one and Western blot and electrophysiology (differences in both Kir6.2 and SUR2A levels) on the other side could be explained by the fact that RT-PCR measured levels of total SUR2A and Kir6.2 mRNA, while Western blot, directly, and patch clamp electrophysiology, indirectly, selectively measured levels of those SUR2A and Kir6.2 subunits that are physically associated to form the channels.
The consequence of the gender-specific difference in cardiac KATPchannel expression has yet to be fully understood. In principle, female gender is associated with a lower incidence of cardiovascular diseases (1–3), and the overexpression of Kir6.2 and SUR2A subunits confers resistance against metabolic stress in an otherwise metabolic stress-sensitive cell line (23,24). From this point of view, it is possible that more cardiac KATPchannels in female tissue than in male tissue would contribute to the higher resistance of females to ischemic heart disease. For example, it has been demonstrated that cardiomyocytes isolated from female hearts are more resistant to severe metabolic stress than those from male hearts (4). In this regard, it has been shown that physiological replacement of estradiol protects the myocardium after global ischemia in the ovariectomized animals (25). In agreement with this concept, we have demonstrated here that cardiomyocytes from female cells are more resistant to ischemia-reperfusion-induced Ca2+loading compared with cardiomyocytes from male tissue. Bearing in mind the difference between estrogen plasma levels in men and women (approximately 4 pg/ml in men vs. approximately 8 pg/ml in women [26,27]), these results are compatible with the notion that estrogens possess direct cardioprotective properties and associate expression of KATPchannels with the estrogen levels.
In this study, RT-PCR analysis did not show differences in Kir6.2 levels between genders, suggesting that a higher number of KATPchannels on sarcolemma are due solely to a difference in SUR2A expression. The presence of more KATPchannels as a consequence of more SUR2A subunits is possible only when the levels of SUR2A in cardiomyocytes are considerably lower then the levels of Kir6.2, that is, a subunit that is less expressed in cardiac tissue controls the number of functional channel proteins. To test the possibility that Kir6.2 is present in higher amounts than SUR2A, we compared RT-PCR products using the primers for Kir6.2 and SUR2A and different amounts of cDNA. Because the PCR reaction was performed under the best conditions for both primers, while all other parameters of PCR reaction were identical, it is plausible to accept that the intensity of PCR-product bands would be a reflection of the SUR2A and Kir6.2 mRNA amounts (28). Reverse transcription polymerase chain reaction analysis demonstrated that SUR2A mRNA is present in considerably smaller amounts than Kir6.2 mRNA, which explains how it is possible that a difference in SUR2A alone is sufficient to create the differences observed in the number of cardiac KATPchannels. In this regard, this is the first report to demonstrate that SUR2A subunit expression is the rate-limiting factor in the production of cardiac KATPchannels.
Conclusion and significance
In conclusion, this study has demonstrated: 1) that female tissue expresses higher levels of functional cardiac KATPchannels than male tissue and this is due to the higher expression of SUR2A subunit; and 2) that the observed difference may underlie the gender-specific difference in susceptibility toward heart diseases. This is the first study that demonstrated a gender-specific difference in cardiac KATPchannel expression as well as the functional importance of this difference. The obtained results could provide a basis for developing therapeutic strategies against ischemic heart disease centered around KATPchannels that would take into account gender-specific differences.
The only limitation of the study is that it was done on guinea pig and not human ventricular tissue and cells. However, bearing in mind similarities between cardiovascular systems in guinea pigs and humans, it is likely that the findings from this study pertain to humans as well.
☆ Supported by grants from the American Heart Association, Biotechnology and Biological Sciences Research Council, British Heart Foundation, National Heart Research Fund, TENOVUS-Scotland and the Wellcome Trust to Dr. Jovanovic.
- complementary DNA
- ATP-sensitive K+
- messenger RNA
- polymerase chain reaction
- reverse transcription polymerase chain reaction
- Received December 15, 2000.
- Revision received May 16, 2001.
- Accepted May 22, 2001.
- American College of Cardiology
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