Chronic Mild Hypoxia Protects Heart-derived H9c2 Cells against Acute Hypoxia/Reoxygenation by Regulating Expression of the SUR2A Subunit of the ATP-sensitive K+ Channel
2003; Elsevier BV; Volume: 278; Issue: 33 Linguagem: Inglês
10.1074/jbc.m303051200
ISSN1083-351X
AutoresRussell M. Crawford, Aleksandar Jovanović, Grant R. Budas, Anthony M. Davies, Harish Lad, Roland H. Wenger, Kevin A. Robertson, Douglas Roy, Harri Ranki, Aleksandar Jovanović,
Tópico(s)Cardiac Arrest and Resuscitation
ResumoChronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO2) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca2+ loading in heart-derived H9c2 cells. The number of ATP-sensitive K+ (KATP) channels were increased in cells exposed to PO2 = 100 mm Hg relative to cells exposed to PO2 = 144 mm Hg. This was due to an increase in transcription of SUR2A, a KATP channel regulatory subunit, but not Kir6.2, a KATP channel poreforming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1α and dominant negative HIF-1β suggested that the HIF-1-signaling pathway did not participate in observed PO2-mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO2 = 100 mm Hg but not the effect of PO2 = 20 mm Hg. LY 294002 and PD 184 352 prevented PO2-mediated regulation of KATP channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO2 = 100 mm Hg, and a decrease of PO2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1α-independent signaling cascade leading to an increase in SUR2A protein, a higher density of KATP channels, and a cellular phenotype more resistant to acute metabolic stress. Chronic exposure to lower oxygen tension may increase cellular resistance to different types of acute metabolic stress. Here, we show that 24-h-long exposure to slightly decreased oxygen tension (partial pressure of oxygen (PO2) of 100 mm Hg instead of normal 144 mm Hg) confers resistance against acute hypoxia/reoxygenation-induced Ca2+ loading in heart-derived H9c2 cells. The number of ATP-sensitive K+ (KATP) channels were increased in cells exposed to PO2 = 100 mm Hg relative to cells exposed to PO2 = 144 mm Hg. This was due to an increase in transcription of SUR2A, a KATP channel regulatory subunit, but not Kir6.2, a KATP channel poreforming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1α and dominant negative HIF-1β suggested that the HIF-1-signaling pathway did not participate in observed PO2-mediated regulation of SUR2A expression. On the other hand, NADH inhibited the effect of PO2 = 100 mm Hg but not the effect of PO2 = 20 mm Hg. LY 294002 and PD 184 352 prevented PO2-mediated regulation of KATP channels, whereas rapamycin was without any effect. HMR 1098 inhibited the cytoprotective effect of PO2 = 100 mm Hg, and a decrease of PO2 from 144 to 100 mm Hg did not change the expression of any other gene, including those involved in stress and hypoxic response, as revealed by Affymetrix high density oligonucleotide arrays. We conclude that slight hypoxia activates HIF-1α-independent signaling cascade leading to an increase in SUR2A protein, a higher density of KATP channels, and a cellular phenotype more resistant to acute metabolic stress. A chronic lack of oxygen has been implicated in variety of diseases including atherosclerosis, diabetes, pulmonary fibrosis, neurodegenerative disorders, arthritis, and aging. At the cellular level, hypoxia activates numerous major signaling pathways, resulting in changes in gene expression, which influence the cellular ability to survive or die. These pathways exert their phenotypic influences largely through modulation of transcription factor activities that effect changes in the pattern of gene expression, and some of these pathways are linked to enhanced survival, whereas others are associated with cell death. Severe hypoxia, occurring at partial pressure of oxygen (PO2) 1The abbreviations used are: PO2, partial pressure of oxygen; GFP, green fluorescent protein; HIF, hypoxia-inducible factor; HPLC, high performance liquid chromatography; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK1, mitogen-activated protein kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C/EBP, CCAAT/enhancer binding protein; AP-1, activator protein-1; mTOR, mammalian target of rapamycin.1The abbreviations used are: PO2, partial pressure of oxygen; GFP, green fluorescent protein; HIF, hypoxia-inducible factor; HPLC, high performance liquid chromatography; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK1, mitogen-activated protein kinase kinase; PI 3-kinase, phosphatidylinositol 3-kinase; RT, reverse transcription; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; C/EBP, CCAAT/enhancer binding protein; AP-1, activator protein-1; mTOR, mammalian target of rapamycin. below 20 mm Hg, impairs cellular energy production and ion homeostasis, leading to cell injury and cell death. In contrast, a lower degree of hypoxia, defined as PO2 between 50 and 100 mm Hg, may activate mechanisms that could produce cellular phenotype more resistant to acute severe oxidative stress (1Martindale J.L. Holbrook N.J. J. Cell. Physiol. 2002; 192: 1-15Crossref PubMed Scopus (1893) Google Scholar, 2Wenger R.H. FASEB J. 2002; 16: 1151-1162Crossref PubMed Scopus (1000) Google Scholar). This phenomenon was in particular described in the heart, where acute severe oxidative stress is one of the most important components of different forms of ischemic heart diseases, including myocardial infarction. At the single cell level, it has been shown that isolated cardiomyocytes when cultured at lower oxygen tension acquire resistance against acute severe oxidative stress (3Silverman H.S. Wei S. Haigney M.C. Ocampo C.J. Stern M.D. Circ. Res. 1997; 80: 699-707Crossref PubMed Scopus (71) Google Scholar). Such single cell reports have been strongly supported by clinical studies showing that the incidence of myocardial infarction complications and the mortality rate are much lower in populations living at lower PO2 than those of the rest of the world (4Ashouri K. Ahmed M.E. Kardash M.O. Sharif A.Y. Abdalsattar M. al Ghozeim A. Ethn. Dis. 1994; 4: 82-86PubMed Google Scholar, 5Hutchison S.J. Litch J.A. J. Am. Med. Assoc. 1997; 278: 1661-1662Crossref PubMed Scopus (13) Google Scholar). How moderate hypoxia induces increased cellular resistance to acute severe oxidative stress is yet unknown. In this regard, the present study was undertaken to address this question and to define the molecular basis of chronic mild hypoxia regulation of cellular resistance to acute metabolic challenges. To achieve this, we applied a set of different techniques on heart-derived H9c2 cells, cells that have been previously successfully implemented to study mechanisms of cellular and cardiac protection (6Ekhterae D. Lin Z. Lundberg M.S. Crow M.T. Brosius F.C. Nunez G. Circ. Res. 1999; 85: e70-e77Crossref PubMed Google Scholar, 7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar). Using this approach we found that chronic minimal hypoxia up-regulates SUR2A subunit of the ATP-sensitive K+ channel without affecting expression of any other gene. This effect is not associated with activation of HIF-1α-dependent signaling pathway, whereas changes in the NAD/NADH ratio and activation of phosphatidylinositol (PI) 3-kinase and extracellular signal-regulated kinases (ERKs) seem to be crucial for the cytoprotective effect of chronic mild hypoxia. A sole increase in SUR2A protein is sufficient to generate more sarcolemmal ATP-sensitive K+ (KATP) channels and create a cellular phenotype resistant to acute severe oxidative stress. Heart H9c2 Cells and Gene Transfection—Rat embryonic heart H9c2 cells (ECACC, Salisbury, UK) 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 some experiments in culture media NADH (20 mm), NAD (20 mm), PD 184352 (10 μm), or rapamycin (1 μm) or LY294002 (50 μm) would be added. For electrophysiological and imaging experiments, the cells were plated on a 35 × 10- or 60 × 15-mm culture dish containing 12- or 25-mm glass cover-slips. The cells were cultured in incubators (Galaxy, oxygen control model, RS Biotech, Irvine, UK) where PO2 was either 144 mm Hg (21% O2, v/v), 100 mm Hg (13% O2), or 20 mm Hg (3%) for 24 h before the experiment. For some experiments H9c2 cells were transfected with HIF-1α (pCMVhHIF-1α) plus HIF-1β/ARNT (pCMVhARNT) or dominant negative ARNT/HIF-1β mutant (pCMVβARNT) or with a ∼1200-bp SUR2 gene promoter fragment flanking the 5′ end of SUR2 gene subcloned in TOPO-Glow vector (Invitrogen; for details see below) and then cultured for 24 h (HIF-1α/HIF-1β or HIF-1β mutant transfected cells were cultured on PO2 = 144 mm Hg and PO2 = 100 mm Hg, respectively, whereas promoter-transfected cells were cultured under both conditions). With HIF-1 subunits and HIF-1β dominant negative subunit, green fluorescent protein (GFP) subcloned into the mammalian expression vector pcDNA3.1+ was routinely cotransfected to enable cell selection for electrophysiology. The cells were transfected with total of 1–2 μg of plasmid DNA at 60–80% confluence using Superfect reagent (Qiagen) according to the manufacturer's instructions. 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, OR). The cells were imaged using a digital epifluorescence imaging system coupled to an inverted microscope (Image Solutions, Standish, UK). A mercury lamp served as a source of light to excite Fura-2AM at 340 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: [Ca2+] = (R – R min/R max – R)K dβ, where R is the fluorescence ratio recorded from the cell, R min and R max are the minimal and maximal fluorescence ratios, K d is 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 in the absence and presence of 100 μm HMR 1098 (Avis Pharma, Frankfurt, Germany) as follows. Single field-stimulated (30 mV, 5 ms, 0.5 Hz) cells were perfused with Tyrode solution containing 136.5 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.53 mm MgCl2, 5.5 mm glucose, 5.5 mm HEPES-NaOH (pH 7.4) at a rate of 1 ml/min. Under these conditions the PO2 in perfusate was ∼140 mm Hg. For hypoxia the solution was continuously bubbled with 100% argon, whereas the exchange of O2 between solution in the chamber and air was prevented by nitrogen jet. The PO2 under these conditions was ∼20 mm Hg. The duration of hypoxia was 10 min, followed by reoxygenation with Tyrode solution for 10 min. Electrophysiology—The cells were superfused with Tyrode solution (136.5 mm NaCl, 5.4 mm KCl, 1.8 mm CaCl2, 0.53 mm MgCl2, 5.5 mm glucose, 5.5 mm HEPES-NaOH, pH 7.4). Pipettes (resistance 3–5 MΩ) were filled with 140 mm KCl, 1 mm MgCl2, 3 mm ATP, 5 mm HEPES-KOH (pH 7.3). The 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 (–100mV to +80 mV in 20-mV steps) were recorded directly to hard disk using an Axopatch-200B amplifier, Digidata-1321 interface, and pClamp8 software (Axon Instruments, Inc., Forster City, CA). 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 currents were low pass filtered at 2 kHz and digitized. Immunoprecipitation and Western Blotting Analysis—Sheep anti-peptide antibodies were raised against synthetic peptides comprised of residues 33–47 in the Kir6.2 protein (ARFVSKKGNCNVAHK) and residues 311–32 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 (10 mm Tris, 20 mm NaH2PO4, 1 mm EDTA, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml pepstatin, 10 μg/ml leupeptin, 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 centrifuged at 500 × g. The supernatant was diluted in buffer II (30 mm imidazole, 120 mm KCl, 30 mm NaCl, 20 mm NaH2PO4, 250 mm sucrose, 10 μg/ml pepstatin, 10 μg/ml leupeptin, pH 6.8) and centrifuged at 7000 × g, the pellet was removed, and the supernatant was centrifuged at 30,000 × g. The obtained pellet contains membrane fraction. The 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 1:1000 dilutions of anti-SUR2A and anti-Kir6.2 antibody, respectively, and detection was achieved using protein G horseradish peroxidase and ECL reagents. Essentially the same protocol was used when Western blot was done with phospho-AP-1 (Abcam, Cambridge), phospho-c-Jun (Abcam), and phospho-C/EBP (Santa Cruz) antibodies on untransfected cells or with anti-GFP antibody (Invitrogen) on cells transfected with the GFP construct containing putative SUR2 promoter fragments (see below); just the total proteins, instead of immunoprecipitates, obtained from H9c2 cells were used, and dilutions of these antibodies were 1:300 to 1:500. RT-PCR—Total RNA was isolated using a commercial kit (RNeasy, Mini Kit, Qiagen) according to the manufacturer's instructions. First strand cDNA was synthesized with random hexanucleotides from 1 mg of total RNA using a reverse transcription system kit (Promega, Southampton, UK). PCR was done using ReadyMix Red Tag from Sigma in a thermal cycler Model Phoenix (Helena Biosciences, Sunderland, UK) under the following conditions: for Kir6.2, 94 °C for 3 min, 34 cycles of 94 °C for 0.5 min, 66.1 °C for 0.5 min, and 70 °C for 1 min, and the final extension at 70 °C for 5 min; for SUR2A, the conditions were the same as for Kir6.2 except that number of cycles was 37–49, and the annealing temperature was 66.1 °C. Two different sets of primers were used to verify any found differences. The primers had the following sequences; for the 387-base-long product for rat Kir6.2 (primer 1), sense, 5′-ATGCGCAAGACCACCAGC-3′, and antisense, 5′-TGGCGGGCTGTGCAGAG-3′; for the 255-base-long product for rat Kir6.2 (primer 2), sense, 5′-GCACCAATGTGCCCTGCGTC-3′, and antisense, 5′-CGGGGTGATCACGGCATGCT-3′; for the 375-base-long product for rat SUR2A (primer 1), sense, 5′-CTAGACGCCACTGTCAC-3′, and antisense, 5′-AGAGAACGAGACACTTGG-3′; and for the 251-base-long product for rat SUR2A (primer 2), sense, 5′-GAGTGTCAGACCTGCGCTTCT-3′, and antisense, 5′-GCTGCTCAGCAGGATTGGTCTC-3′. The levels of GAPDH mRNA was also tested using human GAPDH-primers: sense, 5′-CATCACCATCTTCCAGGAGCGA-3′, and antisense, 5′-GTCTTCTGGGTGGCAGTGATGG-3′, the size of the GAPDH product was 341 bp). There were no significant differences in intensity of GAPDH levels between experimental groups. The nature of the PCR product was confirmed by DNA sequencing. These conditions were set based on our preliminary studies that have demonstrated that under these conditions intensity of the PCR product bend is ∼50% of its maximum. The PCR product band intensities were analyzed using Quantiscan software. DNA Microarray Analysis—Total RNA was isolated from cells cultured at PO2 = 144 mm Hg and PO2 = 100 mm Hg as described for the use of Affymetrix microarrays (8Lee C.K. Klopp R.G. Weindruch R. Prolla T.A. Science. 1999; 285: 1390-1393Crossref PubMed Scopus (1269) Google Scholar). Target RNA was prepared by converting 1 μg of RNA into double-stranded cDNA (Superscript Choice System; Invitrogen) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. Biotin-labeled cRNA was synthesized from cDNA by using an RNA transcript labeling kit (Enzo Biochem). After complementary RNA had been fragmented to sizes ranging from 35 to 200 bases by heating (35 min at 95 °C), 10 μg of RNA fragments were hybridized (16 h at 45 °C) to a Rat Genome U34A array (Affymetrix, Santa Clara, CA). After hybridization, chips were automatically washed and stained with streptavidin-phycoerythrin by using a fluidics system. The chips were scanned with a Hewlett Packard GeneArray Scanner. The Affymetrix RG-U34A array contained ∼7,000 rat genes and expressed ∼1,000 sequence tags from UniGene, GenBank™, and the Institute for Genomic Research data bases. Each gene was presented in the array by 20 perfectly matched oligonucleotides and 20 mismatched control probes that contain a single central-base mismatch. Fluorescence intensity was read for each nucleotide to calculate the average signal intensity for each gene by subtracting the intensities of ∼20 perfectly matched oligonucleotides from the intensity of the mismatched nucleotides. All of the calculations were performed using Affymetrix MAS 4.0 algorithm, i.e. all of the arrays were scaled to an overall target intensity of 100 prior to comparative analysis. Groups (PO2 = 144 mm Hg and PO2 = 100 mm Hg) were compared with each other by pair-wise comparison. Using this method, genes that were present and changed in expression by at least 1.4-fold were meant to be identified. High Performance Liquid Chromatography—The cells were rapidly frozen, and 0.73 m trichloroacetic acid was added. The solution was then homogenized and centrifuged. The supernatant was removed and placed in tri-n-octylamine and FREON (50:50, v/v), vortex-mixed, and centrifuged. The supernatant was taken and used for HPLC. A Nova-Pak (Reading, UK) C18 4-μm spherical radii bead, dimension 300 × 3.9 mm inner diameter column was used. A mobile phase consisting of 12% methanol, 1.47 mm tetrabutylammonium phosphate, 73.5 mm KH2PO4, adjusted to pH 4.0. The flow rate was 1.0 ml/min. NADH and NAD were detected at 254 nm (under these conditions retention times were 9.3 min for NAD and 24.0 min for NADH) using a Varien ProStar HPLC work station (Kinesis, Epping, UK). Cloning, Subcloning, and Use of Human SUR2 Gene Promoter—For the human SUR2 promoter analysis, a fragment extending 1200 bases downwards from the translation initiation triplet was cloned from human genomic DNA (Promega). The sense primer was 5′-GACCTTTGCTCATCTCCCATC-3′, and the antisense primer was 5′-TTTCTTCTTATATGGTTTACTCTAA-3′; PCR was done using Promega PCR core system components. In these reactions the magnesium concentration was 1.87 mm, and the PCR mixtures were run at 94 °C for 5 min; 35 cycles of 94 °C for 45 s, 55 °C for 1 min, and 70 °C for 2 min; and a final extension of 70 °C for 5 min. The PCR product was cloned into TOPO-Glow vector using the manufacturer's instructions (Invitrogen). To create a 380-base-long fragment, a 1200-base-long fragment of putative SUR2 promoter PCR product was fragmented by restriction digestion using the internal BglII site (380 bases from the start codon). 380-bp fragment was blunt end-ligated into TOPO vector (Invitrogen). The plasmids were transiently transfected into H9c2 cells using Qiagen Superfect reagent and the manufacturer's protocol (see above), the cells were then incubated for 24 h at 37 °C (at 5% CO2)onPO2 = 144 mm Hg or PO2 = 100 mm Hg, and the RNA was extracted. 1 μg of RNA was treated with 0.5 Kunitz units of DNase (Qiagen RNase-free DNase kit). DNase-treated RNA was used to make cDNA in a total volume of 20 μl. ∼10% of the cDNA was used in a single PCR to analyze the promoter activity using GFP-specific primers, sense primer was 5′-GGTGATGCTACATACGGAA-3′, and the antisense primer was 5′-TACCTGTCGACACAATCTG-3′. The PCR-run conditions for GFP-primers were 94 °C for 3 min; 35 cycles of 94 °C for 0.5 min, 52 °C for 0.5 min, and 70 °C for 1 min; and a final extension of 70 °C for 5 min. The cDNA was also analyzed for the presence of the vector using promoter-specific primers. The obtained data have been analyzed as described under "RT-PCR." Statistics—The data are presented as the means ± S.E., with n representing the number of experiments. The mean values between two groups were compared by the paired or unpaired Student's t test or Rank tests where appropriate. The results for Kir6.2 and SUR2A obtained with RT-PCR for each sample were normalized taking into account that the mean values between more then two groups were compared by the one-way or one-way Rank analysis of variance. All of the statistical tests were done using the SigmaStat program (Jandel Scientific). p < 0.05 was considered statistically significant. Chronic Mild Hypoxia Confers Resistance to Acute Hypoxia/Reoxygenation-induced Ca2 + Loading in H9c2 Cells—Intracellular Ca2+ loading is a reliable on-line parameter of a metabolic condition in mammalian cells, including heart-derived H9c2 cells (7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar). Under control conditions (cells cultured at PO2 = 140 mm Hg), hypoxia/reoxygenation-induced Ca2+ loading in all cells tested, suggesting that this cellular phenotype is sensitive to such an insult (Fig. 1, A and C). In contrast, the same insult produced intracellular Ca2+ loading only in 8.3% of cells chronically exposed to PO2 = 100 mm Hg (Fig. 1, B and C). Chronic Mild Hypoxia Increased the Number of K ATP Channels in Plasma Membrane by Regulating Expression of SUR2A Subunit—It has been previously shown that the density of KATP channels may regulate cellular resistance to oxidative stress (7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar, 9Ranki H.J. Budas G.R. Crawford R.M. Jovanović A. J. Am. Coll. Cardiol. 2001; 38: 906-915Crossref PubMed Scopus (93) Google Scholar). To assess the relative number of functional KATP channels composed of Kir6.2 and SUR2A subunits (7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar, 10Inagaki N. Gonoi T. Clement J.P. Wang C.Z. Aguilar-Bryan L. Bryan J. Seino S. Neuron. 1996; 16: 1011-1017Abstract Full Text Full Text PDF PubMed Scopus (869) Google Scholar) that are present in plasmalemma, H9c2 membrane fraction was immunoprecipitated with anti-Kir6.2 antibody and probed with the anti-SUR2A antibody and vice versa (immunoprecipitated with anti-SUR2A antibody and probed with anti-Kir6.2 antibody; using this approach only those subunits physically associated with each other were measured) (7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar, 9Ranki H.J. Budas G.R. Crawford R.M. Jovanović A. J. Am. Coll. Cardiol. 2001; 38: 906-915Crossref PubMed Scopus (93) Google Scholar). This strategy revealed more than 2-fold higher levels of Kir6.2 and SUR2A proteins in cells cultured at mild hypoxia compared with those cultured at normoxia (Fig. 2; band density, for Kir6.2 11 ± 3 and 19 ± 2 under control conditions and hypoxia, respectively, and for SUR2A 18 ± 2 and 35 ± 5 under control conditions and hypoxia, respectively; p < 0.01 in both cases; n = 5 for each), although at the same time no significant changes were observed in amounts of secondary antibody heavy chain (for Kir6.2 21.6 ± 2.5 and 20.6 ± 3.2 under control conditions and hypoxia, respectively, and for SUR2A 16.2 ± 1.1 and 16.8 ± 1 under control conditions and hypoxia, respectively; p = 0.59 and 0.37 for Kir6.2 and SUR2A, respectively; n = 5 for each). To determine whether changes in the transcriptional activity of Kir6.2 and SUR2 genes underlie changes in number of plasmalemmal KATP channels, we measured Kir6.2 and SUR2A mRNAs using RT-PCR. We designed two separate sets of primers (see the methods), and we tested whether the primers that we designed and RT-PCR could detect differences in mRNA levels. Therefore, we applied RT-PCR with two sets of Kir6.2 and SUR2A primers on slightly different amounts of DNA template using the same number of cycles. These experiments have demonstrated that the primers and conditions we used are capable of detecting less then 2-fold differences in mRNA (Fig. 3) and that >30 amplification cycles used in this study would not lose difference in initial message. Thus, RT-PCR analysis with two different sets of primers for each subunit demonstrated that levels of Kir6.2 mRNA did not change by mild hypoxia (Fig. 4, B and D). In contrast, >2-fold higher levels of SUR2A mRNA in cells exposed to PO2 = 100 mm Hg relative to those exposed to PO2 = 144 mm Hg were found (Fig. 4, A and C). The applied degree of hypoxia did not affect levels of GAPDH mRNA (Fig. 4E).Fig. 3RT-PCR detects relatively small initial Kir6.2 and SUR2A mRNA differences. Shown are the RT-PCR products obtained with two different, independent sets of Kir6.2- and SUR2A-specific primers on H9c2 cells using different dilutions of the same cDNA pool (left panels) and corresponding graphs (right panels) showing amount-bend intensity relationship.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4Chronic mild hypoxia-induced increase in sarcolemmal K ATP channels is solely mediated by increase in SUR2A channel subunit. A–D, RT-PCR products and corresponding graphs obtained with two different sets of Kir6.2-specific (B and D) and SUR2A-specific (A and C) primers from H9c2 cells cultured at PO2 = 144 mm Hg and PO2 = 100 mm Hg. E, RT-PCR products and corresponding graph obtained with GAPDH-specific primers from H9c2 cells cultured at PO2 = 144 mm Hg and PO2 = 100 mm Hg. Each bar represents the mean ± S.E. (n = 2–4). *, p < 0.05.View Large Image Figure ViewerDownload Hi-res image Download (PPT) SUR2 Gene Promoter Is Regulated by Chronic Mild Hypoxia—To examine whether a slight decrease of PO2 would activate transcription of SUR2A, we cloned the putative human SUR2 gene promoter and measured the promoter-driven expression of a reporter gene (GFP). H9c2 cells were transfected with the 1200-bp fragment of the putative human SUR2 promoter subcloned into the GFP promoterless vector. Under these conditions the transcription of GFP was directly dependent upon activity of SUR2 promoter. RT-PCR analysis demonstrated a >2-fold higher level of GFP mRNA in cells cultured at PO2 = 100 mm Hg compared with those cultured at PO2 = 144 mm Hg (Fig. 5, A and B). No difference was observed between GAPDH levels in transfected cells irrespective of PO2 (Fig. 5C). A computer-assisted search using putative human SUR2 gene promoter sequence revealed the presence of binding sites for CCAAT/enhancer binding protein (C/EBP) and activator protein-1 (AP-1) transcription factors. Mild hypoxia increased phosphorylation of AP-1 transcription factors and in particular c-Jun, whereas phosphorylation of C/EBP was not increased (Fig. 5D). The obtained RT-PCR results with promoter were further confirmed at the level of expressed GFP protein. In H9c2 cells transfected with GFP promoterless vector, no signal for GFP on a Western blot using anti-GFP antibody was visualized (Fig. 5E). On the other hand, when constructs containing a 1200- or 380-bp putative promoter fragment were introduced into H9c2 cells, GFP was detected in total protein extract (Fig. 5E). Exposure to mild hypoxia increased the level of expressed GFP regardless of whether cells were transfected with the 1200- or 380-bp putative promoter fragment (Fig. 5E). Chronic Mild Hypoxia-induced Increase in Number of Sarcolemmal K ATP Channels Is Independent on HIF-1α—It is well established that chronic hypoxia regulates genes expression primarily via HIF-1 (11Wang G.L. Semenza G.L. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4304-4308Crossref PubMed Scopus (1176) Google Scholar). To determine whether HIF-1 mediates mild hypoxia-induced increase in the number of KATP channels, we employed patch clamp electrophysiology on untransfected and transfected H9c2 cells exposed to different oxygen tensions. Pinacidil, a prototype KATP channel opener, induces whole cell K+ current proportionally to the number of KATP channels in membrane (7Ranki H.J. Budas G.R. Crawford R.M. Davies A.M. Jovanović A. J. Am. Coll. Cardiol. 2002; 40: 367-374Crossref PubMed Scopus (98) Google Scholar, 9Ranki H.J. Budas G.R. Crawford R.M. Jovanović A. J. Am. Coll. Cardiol. 2001; 38: 906-915Crossref PubMed Scopus (93) Google Scholar, 12Ranki H.J. Crawford R.M. Budas G.R. Jovanović A. Mech. Ageing Dev
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