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

Abstract

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+ (K(ATP)) 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 K(ATP) channel regulatory subunit, but not Kir6.2, a K(ATP) channel pore-forming subunit. PO2 = 100 mm Hg also increased the SUR2 gene promoter activity. Experiments with cells overexpressing wild type of hypoxia-inducible factor (HIF)-1alpha and dominant negative HIF-1beta 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 K(ATP) 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-1alpha-independent signaling cascade leading to an increase in SUR2A protein, a higher density of K(ATP) channels, and a cellular phenotype more resistant to acute metabolic stress.

Fig. 1. Chronic mild hypoxia confers resistance to acute hypoxia/reoxygenation in H9c2 cells

A and B, epifluorescent digital images of cells cultured at PO₂ = 144 mm Hg (A) and PO₂ = 100 mm Hg (B) loaded with Fura-2 prior (Control) and following hypoxia/reoxygenation (Hypoxia/reoxygenation). The white bar corresponds to 30 μm. The graphs are time courses of intracellular concentration of Ca²⁺ (A1 and B1 correspond to A and B, respectively). Each line on the graphs represents a single cell from the corresponding image field. C, percentage of cells cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg that responded (defined as 50% increase in resting Ca²⁺ with the time course pattern of Ca²⁺ increase as depicted in A) or did not respond to hypoxia/reoxygenation with Ca²⁺ loading (n = 12-24). *, p = 0.0006.

Fig. 2. Chronic mild hypoxia induces increase in sarcolemmal K_ATP channels

Shown are Western blots and corresponding graphs with anti-Kir6.2 (A) and anti-SUR2A (B) antibodies of anti-SUR2A (A) and anti-Kir6.2 (B) immunoprecipitate from membrane fractions from H9c2 cells cultured at PO₂ = 144mm Hg and PO₂ = 100 mm Hg. The blots were cross-probed (anti-Kir6.2 antibody was used on anti-SUR2A immunoprecipitate, and anti-SUR2A antibody was used on anti-Kir6.2 immunoprecipitate). Each bar represents the mean ± S.E. of the mean (n = 5 for each). *, p < 0.05. HC, heavy chain.

Fig. 3. RT-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.

Fig. 4. Chronic 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.

Fig. 5. Chronic mild hypoxia regulates activity of human SUR2 promoter

A and B, RT-PCR products and corresponding graphs obtained with GFP-specific primers from H9c2 cells transfected with SUR2 promoter-GFP gene using different amounts of cDNA (A and A1) or with GFP-specific primers from untransfected H9c2 cells and cells transfected with SUR2 promoter-GFP gene cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg (B and B1). Each bar/point represents the mean ± S.E. (n = 2-3). *, p < 0.05. C, RT-PCR products obtained with GAPDH-specific primers from transfected H9c2 cells cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg. D, Western blots with general anti-phospho-AP-1 (AP-1), anti-phospho-c-jun (c-Jun), and anti-phospho-C/EBP (C/EBP) antibodies of total proteins from H9c2 cells cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg. E, Western blots with anti-GFP antibody of total proteins from H9c2 cells transfected with promotorless TOPO-Glow vector (No promoter) TOPO-Glow vector containing 1200 (Promoter (1200 bp)) and 380 (Promoter (380 bp)) bases cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg.

Fig. 6. Chronic mild hypoxia increases pinacidil-induced whole cell membrane current

A, membrane currents evoked by identical families of 400-ms voltage pulses in cells that were first maintained under control conditions and then exposed to 100 μM pinacidil for 2 min and in cells cultured at PO₂ = 144mm Hg and PO₂ = 100 mm Hg. A1 and A2, current-voltage relationships for conditions in A (graphs are aligned with corresponding experiments above). The pinacidil-sensitive component of current (B) for cells in A and current density (C) at 80 mV. Each bar represents the mean ± S.E. (n = 7 for each). *, p < 0.05. The arrowheads indicate zero current levels.

Fig. 7. Chronic mild hypoxia-mediated increase in K_ATP channels is not mediated by HIF-1

A, membrane currents evoked by identical families of 400-ms voltage pulses in cells that were first maintained under control conditions and then exposed to 100 μM pinacidil for 2 min, in cells transfected with HIF-1α/HIF-1β and cultured at PO₂ = 144 mm Hg, and in cells transfected with dominant negative HIF-1β cultured at PO₂ = 100 mm Hg. A1 and A2, current density at 80 mV of pinacidil-sensitive component of current for cells in A (graphs are aligned with corresponding experiments above). Each bar represents the mean ± S.E. (n = 7 for each). The arrowheads indicate zero current levels. B, RT-PCR products obtained with SUR2A-specific primers from H9c2 cells cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg in the absence (C lanes) and presence of 5 μM cadmium (Cd lanes). B1, graph corresponding to RT-PCR products depicted in B. Each bar represents the mean ± S.E. (n = 3).

Fig. 8. Changes in NAD/NADH ratio and activation of PI 3-kinase and ERK are involved in the effect of chronic mild hypoxia

Shown are typical Western blots of anti-Kir6.2 immunoprecipitates from membrane fraction obtained from H9c2 cells and probed with anti-SUR2A antibody under depicted conditions (concentrations used were 20 mM NADH, 20 mM NAD, 10 μM PD 184352, 1 μM rapamycin, and 50 μM LY294002).

Fig. 9. Chronic mild hypoxia protects H9c2 cells against acute hypoxia/reoxygenation by K_ATP channel-dependent mechanism

Shown are epifluorescent digital images of cells cultured at PO₂ = 144 mm Hg (A) and PO₂ = 100 mm Hg (B) loaded with Fura-2 prior (control), and following hypoxia/reoxygenation in the presence of HMR 1098 (100 μM), a selective antagonist of sarcolemmal K_ATP channels. Magnification was ×40. C, percentage of cells cultured at PO₂ = 144 mm Hg and PO₂ = 100 mm Hg that responded (defined as 50% increase in resting Ca²⁺ with the time course pattern of Ca²⁺ increase as depicted in A) or did not respond to hypoxia/reoxygenation with Ca²⁺ loading in the presence of HMR 1098 (n = 12-21).