Disrupting KATP channels diminishes the estrogen-mediated protection in female mutant mice during ischemia-reperfusion
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- Gao, J., Xu, D., Sabat, G. et al. Clin Proteom (2014) 11: 19. doi:10.1186/1559-0275-11-19
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Estrogen has been shown to mediate protection in female hearts against ischemia-reperfusion (I-R) stress. Composed by a Kir6.2 pore and an SUR2 regulatory subunit, cardiac ATP-sensitive potassium channels (KATP) remain quiescent under normal physiological conditions but they are activated by stress stimuli to confer protection to the heart. It remains unclear whether KATP is a regulatory target of estrogen in the female-specific I-R signaling pathway. In this study, we aimed at delineating the molecular mechanism underlying estrogen modulation on KATP channel activity during I-R.
Materials and methods
We employed KATP knockout mice in which SUR2 is disrupted (SUR2KO) to characterize their I-R response using an in vivo occlusion model. To test the protective effects of estrogen, female mice were ovariectomized and implanted with 17β-estradiol (E2) or placebo pellets (0.1 μg/g/day, 21-day release) before receiving an I-R treatment. Comparative proteomic analyses were performed to assess pathway-level alterations between KO-IR and WT-IR hearts.
Results and discussion
Echocardiographic results indicated that KO females were pre-disposed to cardiac dysfunction at baseline. The mutant mice were more susceptible to I-R stress by having bigger infarcts (46%) than WT controls (31%). The observation was confirmed using ovariectomized mice implanted with E2 or placebo. However, the estrogen-mediated protection was diminished in KO hearts. Expression studies showed that the SUR2 protein level, but not RNA level, was up-regulated in WT-IR mice relative to untreated controls possibly via PTMs. Our antibodies detected different glycosylated SUR2 receptor species after the PNGase F treatment, suggesting that SUR2 could be modified by N-glycosylation. We subsequently showed that E2 could further induce the formation of complex-glycosylated SUR2. Additional time-point experiments revealed that I-R hearts had increased levels of N-glycosylated SUR2; and DPM1, the first committed step enzyme in the N-glycosylation pathway. Comparative proteomic profiling identified 41 differentially altered protein hits between KO-IR and WT-IR mice encompassing those related to estrogen biosynthesis.
Our findings suggest that KATP is likely a downstream regulatory target of estrogen and it is indispensable in female I-R signaling. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be effective to enhance KATP channel subunit expression in I-R.
KeywordsKATP channel Sulfonylurea receptor Myocardial infarction Gender difference Estrogen Estrogen receptor Glycosylation
Myocardial infarction (MI) is a life-threatening event that can cause sudden cardiac arrest in patients; and those who survive the first MI likely have repeated incidences and develop heart failure . During the on-set of MI, cardiac cells die within minutes from insufficient blood supply and oxygen, leading to irreversible injuries to the myocardium. Existing epidemiological data reveals that pre-menopausal women have a relatively lower risk of MI than age-matched men [2, 3]. For example, MI incidences occurred in females are only 1/3 of their male counterparts in the 35–44 and 45–54 years-old groups . However, this “female advantage” diminishes upon aging as estrogen level declines, supporting the notion that estrogen is a key modulator in mediating protection to a female heart [2, 3]. Estrogen exerts its effects by binding to estrogen receptors (ER), ERα and ERβ, to regulate the downstream targets . Both ER subtypes are detected in various cardiac cellular compartments encompassing nucleus, plasma membrane and mitochondria [6, 7]. These receptors are likely integral members of the female cardioprotective network but they may govern different signal transduction pathways [8, 9].
ATP-sensitive potassium channels (KATP) are known to play a pivotal role in conferring protection to the heart . These channels remain closed under normal physiological conditions but they open in response to cellular stress such as ischemia. The opening of KATP channels is thought to re-polarize cardiac cell membrane and reduce calcium loading to the heart . Sarcolemmal KATP channels primarily contain a Kir6.2 pore  and a sulfonylurea receptor 2 (SUR2) regulatory subunit [13, 14, 15], where SUR2 regulates the pore activity and channel kinetics. More recent studies have shown that KATP density in plasma membrane is significantly higher in female than male cardiomyocytes , suggesting that female hearts may possess higher KATP activity. Administrating KATP blockers to both genders of mice before an ischemic insult readily diminishes the female advantage against ischemia-reperfusion (I-R) stress . In aged female myocytes, however, KATP channel density dramatically declines but remains unchanged in aged male cells . These observations have provided a basis for estrogen modulation on KATP activity but the underlying mechanism is not fully understood.
In this report, we characterized the I-R response in SUR2 knockout (SUR2KO) female mice using a combined approach of ovariectomized models and comparative proteomics. Our findings identified KATP as a downstream regulatory target of estrogen and it plays a critical role in the mechanistic pathway of female stress signaling.
Results and discussion
SUR2KO female mice display cardiac dysfunction at baseline
SUR2KO females have larger infarcts post ischemia-reperfusion
The protective effect of estrogen is diminished in SUR2KO females
qRT-PCR data detected no significant differences in SUR2 transcripts between WT-E2 and WT-placebo hearts post I-R (Figure 3D). Western blot results showed that WT-E2 mice had a 6-fold higher SUR2 level than WT-placebo mice (Figure 3E). The data suggested that the increased SUR2 protein expression might occur at the post-translational level, which was mediated by estrogen. This observation agreed with a previous report showing that E2-ERβ action can induce S-nitrosylation, a PTM mechanism, to up-regulate levels of a subset of proteins with protective properties .
SUR2 is modified by N-glycosylation
Dolichol monophosphate mannose synthase (DPM1) catalyzes the first committed-step reaction in the N-glycosylation pathway . DPM1 is responsible to transfer mannose from GDP-mannose to dolichol monophosphate to form dolichol monophosphate mannose as the mannosyl donor during N-glycosylation. Expression level of DPM1 was previously reported to be markedly enhanced by estrogen in mouse uteri . In this experiment, we compared DPM1 levels in I-, R- and IR- treated mice. DPM1 level was increased by 4.5- or 3-fold in the I-R or I group relative to the R group (Figure 5B), indicating that the general capacity of N-glycosylation was also up-regulated in ischemic female hearts.
SUR2 N-glycosylation is mediated by estrogen
Estrogen has been shown to increase membrane density of KATP channels in cultured H9c2 cells . We tested whether E2 could further induce the glycosylated SUR2 receptor species using a heterologous expression system. A full-length SUR2 cDNA was introduced into a COS1 cell line that has a stably expressed Kir6.2 pore . 24 h post transfection, cells were incubated with 100 nM E2 or DMSO (vehicle) for 24 h. When T1 was used to probe the samples, the core-glycosylated 140-kDa SUR2 was the major species in the DMSO-treated control cells. In the E2-treated cells, however, the complex-glycosylated 150-kDa SUR2 was the major species (Figure 5C). The data suggested that estrogen could further induce the complex-glycosylated SUR2. This species likely confers the glybenclamide-sensitive KATP currents in our earlier studies [19, 20].
SUR2KO Proteomic Changes Post I-R
Protein hits that are differentially expressed in SUR2KO-IR and WT-IR hearts
Fold of change (KO/WT)
Mitochondrial peptide methionine sulfoxide reductase
40S ribosomal protein S3
Estradiol 17-beta-dehydrogenase 10
Estradiol 17-beta-dehydrogenase 8
Dynamin-like 120 kDa protein
Mitochondrial 2-oxoglutarate/malate carrier protein
ATP synthase subunit gamma, mitochondrial
ATP synthase subunit alpha, mitochondrial
Cytochrome c oxidase subunit 5A, mitochondrial
Propionyl-CoA carboxylase beta chain, mitochondrial
Heat shock protein HSP 90-beta
Cytochrome b-c1 complex subunit 1, mitochondrial
CDGSH iron-sulfur domain-containing protein 1
T-complex protein 1 subunit beta
Basement membrane-specific heparan sulfate proteo-glycan core protein
Pyruvate dehydrogenase protein X component, mitochondrial
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13
Succinate dehydrogenase [ubiquinone] iron-sulfur subunit, mitochondrial
Electron transfer flavoprotein subunit beta
ATP synthase subunit d, mitochondrial
Hemoglobin subunit beta-1
Cytochrome b-c1 complex subunit 8
Ferritin light chain 1
Acyl-CoA dehydrogenase family member 10
NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 7
Laminin subunit gamma-1
AFG3-like protein 2
Glutathione S-transferase P 1
NSFL1 cofactor p47
Ubiquitin carboxyl-terminal hydrolase 14
Vesicle-associated membrane protein-associated protein B
Adenylyl cyclase-associated protein 1
Only detected in KO
Our physiological data (Figures 2 and 3) showed that SUR2KO female mice resembled those aged WT females in prior reports [2, 3, 4], which display a compromised estrogenic effect and cardioprotection. In our proteomic study, levels of estradiol 17β-dehydrogenase 8 (Hsd17β8) and 10 (Hsd17β10) were found to be significantly decreased by 2-folds in KO-IR mice. Hsd17β-based enzymes belong to the short-chain dehydrogenase/reductase superfamily. They are mainly involved in the biosynthesis of estrogens, androgens and fatty acids . Hsd17β8 catalyzes the inter-conversion between E2 (estradiol) and E1 (estrone). E2 is the predominant form of circulating estrogen before menopause while E1 is the major estrogen type present in the postmenopausal stage . Hsd17β8 primarily acts as an oxidative enzyme to inactivate E2. However, it has some reductase activity and can produce E2 from E1. Thus, Hsd17β8 is an important Hsd17β member that controls concentration of estrogen in the cell. It seems that KO-IR female mice are “locked” into a relatively high E1 state, mimic the postmenopausal women group. The high E1 level may auto-suppress the activity of Hsd17β8, which prevents further inactivation of E2 or over-accumulation of E1. Our results therefore provided first line of evidence that disrupting KATP channels affects estrogen biosynthesis in mice. The loss of KATP as a downstream target for E2 modulation likely affects other related molecular signaling pathways (Additional file 2). On the other hand, Hsd17β10 mainly functions in the mitochondria to catalyze beta-oxidation at position 17 of estrogen or androgen. An earlier study has reported that Hsd17β10 deficiency caused by genetic mutations can result in Alzheimer’s disease . In addition to the findings in estrogen-related targets, we found hits (Lamc1, Hspg2 and LPL) that are related to N-glycosylation in our GO study. Levels of these three proteins were found to be higher in the KO-IR hearts. A previous study showed that level of N-glycosylation in Lamc1 or Hspg2 was altered in I-R rat hearts . Changes in these proteins could be related to the increase of general N-glycosylation capacity in I-R. We also noticed that levels of certain targets related to actin cytoskeleton were altered. The alteration may be associated with the detected hypertrophy in KO hearts.
More recent studies have shown that KATP channels are also present in the inner membranes of mitochondria . We previously reported that the diazoxide-sensitive mitochondrial KATP activity was absent in SUR2KO mice . In our proteomic study, we found that 50% of the identified protein hits are associated with mitochondrial function and energy generation (Table 1). The finding revealed that the KO female mitochondria were severely altered in I-R. One important hit, optic atrophy 1 (OPA1), is known to play a key role in shaping the mitochondria and fusion to the inner membrane . A recent report shows that a decreased level of OPA1 is associated with heart failure . We detected a significant 2-fold reduction in OPA1 level in SUR2KO-IR hearts, suggesting that disrupting KATP likely affects OPA1 expression and the fusion/fission rate of mitochondria. It is known that estrogen in involved in modulating mitochondrial biogenesis and maintaining mitochondrial membrane potential . Functional estrogen receptor, ERβ, has been found in the mitochondria  to mediate anti-apoptotic response  and cardioprotection [8, 25]. The large numbers of mitochondrial hits identified in our proteomic study may be due to a combined effect from losing the diazoxide-sensitive mitochondrial KATP activity and altered mitochondrial ERβ level. Future compartment-specific proteomic analyses using enriched fractions isolated from mitochondrial or plasma membranes may identify additional targets that are related to estrogen regulation and female-specific stress signaling network.
The regulatory subunit for the cardiac KATP channels, SUR2, is a low-affinity sulfonylurea receptor . Because sulfonylureas are commonly used in treating diabetes mellitus, knowledge about SUR2 regulation by estrogen is expected to shed new light into hormonal regulation of a “cardioprotective” ion channel and the related molecular signaling pathways. Modulating activities of key ion channels via targeting their regulatory subunits has been employed as a new therapeutic strategy to treat certain cardiovascular diseases. Increasing SUR2 expression by N-glycosylation mediated by estrogen may be an effective manner to enhance KATP channel density in the heart. The novel finding that KATP is a downstream regulatory target of estrogen will provide new perspectives in future estrogen replacement therapies to postmenopausal women. The different I-R response in both genders of SUR2KO mice indicates that these models are innovative platforms to study gender-specific divergence in other cardiovascular diseases.
Mouse protocols and handling were performed following the guidelines of National Institutes of Health. All mice were maintained at the University of Wisconsin Animal Core Facility. SUR2 knockout mice were previously created by inserting a disruption cassette at exons 12–16 of the SUR2 gene . C57BL-6 J mice (Jackson Laboratories, Bar Harbor, Maine) that were heterozygous for the Sur2 locus were bred into the FVB background . Heterologous mice were interbred and genotyped to obtain homozygous mutants. Age-matched female SUR2KO and WT littermates were used in this study.
SUR2 antibodies were custom-designed antibodies that were generated as previously described . Anti-GAPDH was obtained from Assay Designs (Ann Arbor, MI); anti-ERα was from Santa Cruz; anti-ERβ was from Millipore; anti-concanavalin A was from Vector Laboratories (Burlingame, CA) and anti-DPM1 was from IMGENEX (San Diego, CA). Secondary antibodies were obtained from GE Healthcare (Piscataway, NJ).
Mice were lightly anesthetized before echocardiographic recordings using Vevo Model770 (VisualSonics, Canada). Isoflurane was delivered at 3% during induction and at 1% for maintenance via nose cone. Images were captured using a 40-mHz mechanical transducer.
An ischemia-reperfusion (I-R) protocol operated as previously described with modifications [22, 23]. An open-chest occlusion model was used to induce I-R in SUR2KO and WT female mice. Mice were induced with 2% isoflurane, intubated and ventilated at 150 breaths/min at 200–300 μL tidal volume. Body temperature was maintained with a heating pad. Electro-cardiogram (ECG) was used to monitor mice and the ECG lead was processed with a Gould amplifier and digitally converted for off-line analysis. The protocol included a 30-min ischemia phase followed by a 90-min reperfusion period. Epicardial cyanosis, alteration in myocardial contractility, and ST segment elevation were used to confirm ischemia. Reperfusion was initiated by unclamping the hemostat and loosening the suture from the polyethylene tubing, and it was confirmed by elimination of epicardial cyanosis and normalization of the S-T segment. Infarct sizes were determined as previously described .
Estrogen delivery in mice
Adult WT or SUR2KO female mice at the age of 9 wks were subject to ovariectomization . These mice were subsequently implanted with 17β-estradiol (E2) or placebo pellets (0.1 μg/g/day, 21-day release, Innovative Research of America, Sarasota, FL). All mice received our I-R protocol on Day 18-20.
Total RNA was isolated from LV tissues using TRIzol reagents (Life Technologies) and RT-PCR reactions were carried out as previously described . Primers used to amplify SUR2A are: 5’-TGGTGGTACCTCACTTCAGGA-3’ and 5’-CAGGATGGTTTATACTGTA- TTCGGA-3’. Controls primers used to amplify GAPDH are: 5’-AGACATCTAAGGTT- CCAGTATGAC-3’ and 5’-ATCGTCCCATTTGATGTTAGAG-3’. Banding density was scanned by the UVP BioSpectrum Imaging System (Upland, CA) and normalized to GAPDH.
Protein extraction and Western blot analysis
LV tissues were carefully isolated for protein extraction and concentrations were determined using a DC Protein Assay Kit (Bio-Rad, Hercules, CA) as previously reported. Protein samples were separated on 4-12% MOPS NuPAGE gels unless stated elsewhere. Western blots were performed more than three times. Chemiluminescence was detected using an ECL-Plus Detection Kit (GE Healthcare). Blots were scanned and banding densities were determined by the UVP Imaging System.
Mouse LV tissues were isolated from 10–12 wks-old female hearts and subjected to membrane protein isolation. Plasma membrane proteins were purified from mouse LV or cell lysates using a MEM-PER Eukaryotic Membrane Protein Extraction kit (Thermo). 300 μg LV membrane proteins were treated by an enzymatic de-glycosylation reaction using a peptide N-glycosidase F (PNGase F) following manufacturer’s protocol (Sigma, St. Louis, MO). Protein samples were ran on 3-8% Tris-acetate NuPAGE gels to allow better separation. The blots were probed with anti-SUR2 (T1) at 1:2000 or BNJ-2 at (1:1000).
Estrogen treatment in cultured cells
For COS1 cell culture, cells were seeded on a 35-mm-diameter plate (1×105) containing complete MEM medium (Life Technologies), 10% fetal bovine serum, 2 mM L-glutamine, 0.1 nM MEM non-essential amino acid solution, 1 mM MEM pyruvate solution, 10 U penicillin and 10 g streptomycin. SUR2A was co-transfected into a COS1 line stably expressed a Kir6.2 pore  using a TransIT-COS transfection kit (Mirus, Madison WI). 24 h post transfection, cells were treated with 100 nM E2 or DMSO (vehicle) for 24 h. Cells were subjected to membrane protein isolation for Western blot analysis.
Co-IP experiments were carried out using a Classic Co-IP Kit (Thermo) following manufacturer’s recommended procedures. 7 μg anti-SUR2 (T1) was used to IP 500 μg LV proteins (combined from five independently handled mice) from R-, I- or IR- mice followed by immunoblotting using an anti-ConA.
Isolated LV tissues from three independently handled SUR2KO-IR or WT-IR mice were used for protein extraction in the presence of one protease cocktail inhibitor tablet (Roche, Indianapolis, IN). 600 μg protein from each sample was subjected to an albumin depletion column (Qiagen) to remove excessive albumin. “In-liquid” digestion and subsequent mass spectrometric analysis were carried out at the University of Wisconsin Mass Spectrometry Facility. 200 μg of albumin-depleted protein sample was re-solubilized and denatured in 15 μL buffer containing 8 M urea, 50 mM NH4HCO3 (pH8.5) and 1 mM Tris–HCl (pH7.5) for 10 min at room temperature. The mixture was diluted to 60 μL in the reduction/ alkylation step by adding 2.5 μL of 25 mM DTT, 5 μL MeOH and 37.5 μL of 25 mM NH4HCO3 (pH8.5). The mixture was incubated at 50°C for 15 min. Once it was cooled down to room temperature, 3 μL of 55 mM iodoacetamide was added and the mixture was incubated in the dark at room temperature for 15 min. The reaction was quenched by adding 8 μL of 25 mM DTT. Peptides were released using 30 μL Trypsin Gold solution (100 ng/μL in 25 mM NH4HCO3, Promega, Madison, WI) to reach a 100-μL final volume. Digestion was conducted at 42°C for 1 h. Then 15 μL of fresh trypsin solution was added to reach a final enzyme:substrate ratio of 1 to 44, and the digestion reaction was carried out at 37°C for overnight. The reaction was terminated by adding 2.5% trifluoroacetic acid to reach a 0.3% final concentration. 8 μL (12 μg) of the peptide mixture was loaded for subsequent mass spectrometry analysis.
Mass spectrometry procedure
Peptides were analyzed by Nano-LC-MS/MS using the Agilent 1100 NanoFlow System (Agilent Technologies, Santa Clara, CA), which was connected to a hybrid Linear Ion Trap-OrbiTrap Mass Spectrometer (LTQ-OrbiTrap ) equipped with a Nano-electrospray ion source (Thermo-Fisher Scientific). HPLC was performed using a 15-cm column packed with MAGIC C18AQ 3-μm 200 Å particles (MICHROM BioResources, Auburn, CA) and a P-2000 laser pulled tip (Sutter Instrument, Novato, CA) that was connected with a 360 μm × 75 μm fused silica tubing. Sample loading and desalting were carried out at a 10 μL/min rate using a Zorbax 300SB-C18 trapping column (Agilent Technologies), which was in line with an auto-sampler. Peptide elution was carried out using solvents comprised of 0.1% formic acid in water (Solvent A) and 0.1% formic acid/95% acetonitrile in water (Solvent B). The gradient consisted of a 20-min loading and desalting period with a stepwise column equilibration at 1% Solvent B initially, ramp to 40% B for 195 min, ramp to 60% B for 20 min, ramp to 100% B for 5 min and held for 3 min. The column was then re-equilibrated at 1% Solvent B for 30 min. The flow rate for peptide elution and re-equilibration was 0.2 μL/min.
The LTQ-Orbitrap was set to acquire MS/MS spectra in a data-dependent mode. MS survey scans from m/z 300 to 2000 were collected in centroid mode at a resolving power of 100,000. In each scan, MS/MS spectra were collected from the five most-abundant output signals. Dynamic exclusion was employed to increase dynamic range and maximize peptide identification. This feature excluded precursors up to 0.55 m/z below and 1.05 m/z above those previously selected precursors. Precursors remained on the exclusion list for 40 s. Singly-charged ions or ions for which the charge state could not be assigned were rejected for further consideration.
Raw MS/MS data was searched against the UniProt Mouse Amino Acid Sequence Database, which contains over 30,634 protein entries. A Mascot Search Engine 2.2.07 (Matrix Science, Boston, MA) was used in the search with fixed cysteine carbamidomethylation, variable methionine oxidation and asparagine/glutamine deamidation. Peptide mass tolerance was set at 20 ppm and fragment mass at 0.8 Dalton. Protein annotation, significance of identification and spectral based quantification were carried out using the Scaffold Software Package 4.1.1 (Proteome Software, Portland, OR). In this version, quantitative values of protein hits were normalized to total spectral counts, enhancing the accuracy in target identification. Samples from three independently handled KO-IR or WT-IR mice were analyzed. Protein probabilities were assigned by the Protein Prophet algorithm . An average of 494 hits was identified in each sample. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least two identified peptides. Among the total 612 protein hits found from the three sets of samples, the prophet false discovery rate (FDR) for proteins was at 0.1%. Peptide identifications were accepted if they could be established at greater than 90.0% probability as specified by the Peptide Prophet algorithm . Based on the ~66,000 spectra that were found from the three sets of samples, the prophet FDR for spectra was at 0.9%. Proteins that contained similar peptides and could not be differentiated based on MS/MS analysis alone were grouped to satisfy the principles of parsimony.
Pathway enrichment analysis
Using the normalized spectrum data collected from KO-IR or WT-IR mice, Gene Ontology [39, 40] and pathway enrichment [41, 42] were performed using the DAVID (Database for Annotation, Visualization and Integrated Discovery) Bioinformatics Resources v6.7 (NIAID, NIH). DAVID categorizes genes based on enrichment of GO classification, Kyoto Encyclopedia of Genes and Genomes (KEGG), and information from other databases. P-values were used to report whether significance of overlaps with known functional categories was found.
Other statistical analysis
Data were reported as mean ± SEM in the echocardiography and I-R experiments while other data were reported as mean ± STDEV. Statistical analysis was performed using Origin Version9. Statistical significance was determined by Student t test (2-tailed) for two experimental groups or one-way ANOVA for multiple groups with post hoc test using the Bonferroni method. A p < 0.05 was considered statistically significant.
We sincerely thank Dr. Elizabeth McNally and Dr. Jonathan Makielski for providing the SUR2 knockout mouse model and antibodies to this work. We also thank Dr. Timothy Hacker and Dr. Guo-Qing Song for their excellent technical assistance. This work was supported by funding from NIH-NHLBI and NIH Office of Research on Women’s Health (HL-93626), The American Heart Association National Center (0630268N), and The American Heart Association Midwest Affiliation (11GRNT7600070) to NQS. WX is a DOD “Era of Hope Scholar” (W81XWYH-11-1-0237).
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