Mild hypoxia in vivo regulates cardioprotective SUR2A: A role for Akt and LDH

Abstract

High-altitude residents have lower mortality rates for ischaemic heart disease and this is ascribed to cardiac gene remodelling by chronic hypoxia. SUR2A is a cardioprotective ABC protein serving as a subunit of sarcolemmal ATP-sensitive K(+) channels. The purpose of this study was to determine whether SUR2A is regulated by mild hypoxia in vivo and to elucidate the underlying mechanism. Mice were exposed to either 21% (control) or 18% (mild hypoxia) oxygen for 24h. Exposure to 18% oxygen did not affect partial pressure of O(2) (PO(2)) and CO(2) (PCO(2)) in the blood, haematocrit or level of ATP in the heart. However, hypoxia increased myocardial lactate dehydrogenase (LDH) and lactate as well as NAD(+) without affecting total NAD. SUR2A levels were significantly increased as well as myocardial resistance to ischaemia-reperfusion. Exposure to 18% oxygen did not phosphorylate extracellular signal regulated kinases (ERK1/2) or AMP activated protein kinase (AMPK), but it phosphorylated protein kinase B (Akt). An inhibitor of phosphoinositide 3-kinases (PI3K), LY294002 (0.2mg/mouse), abolished all observed effects of hypoxia. LDH inhibitors, galloflavin (50 μM) and sodium oxamate (80 mM) significantly decreased levels of SUR2A in heart embryonic H9c2 cells, while inactive mutant LDH form, gly193-M-LDH increased cellular sensitivity towards stress induced by 2,4-dinitrophenol (10mM). Treatment of H9c2 cells with sodium lactate (30 mM) increased intracellular lactate, but did not affect LDH activity or SUR2A levels. We conclude that PI3K/Akt signalling pathway and LDH play a crucial role in increase of cardiac SUR2A induced by in vivo exposure to 18% oxygen.

Keywords: Akt; Heart; Hypoxia; LDH; Oxygen; SUR2A.

Fig. 1

Western blot signals obtained by anti-AMPK antibodies were of appropriate molecular weight and clearly distinguishable from any other signals appearing on blots. Typical examples of original Western blots in this study when using anti-AMPK (AMPK) and anti-phospho-AMPK (P-AMPK) antibodies.

Fig. 2

Systemic exposure to 18% oxygen regulates levels of NAD +, NADH, lactate, LDH and SUR2A in the heart. Bar graphs depicting PO₂ (O₂), PCO₂ (CO₂) and haematocrit (HCT) in the blood as well as ATP (ATP), lactate (Lactate), LDH, total NAD (NADt), NADH (NADH), NAD + (NAD +) and SUR2A (SUR2A) in the myocardial tissue; inset in SUR2A bar graph represents original Western blots for SUR2A under labelled conditions. Each bar is a mean ± SEM (n = 3–5). *P < 0.01. AU = arbitrary units.

Fig. 3

Exposure to 18% oxygen increases heart resistance to ischaemia–reperfusion. Bar graphs depicting size of myocardial infarction, CK and LDH concentration in heart effluent following ischaemia–reperfusion. Each bar is a mean ± SEM (n = 3–5). *P < 0.05.

Fig. 4

Systemic exposure to 18% oxygen does not phosphorylate ERK1/2 in the heart. Original Western blots with phospho-ERK1, phospho-ERK2 and total ERK1 and ERK2 antibodies applied on extracts from hearts under depicted conditions and corresponding graphs. Each bar is a mean ± SEM (n = 4-5). AU = arbitrary units.

Fig. 5

Systemic exposure to 18% oxygen does not phosphorylate AMPK in the heart. Original Western blots with phospho-AMPK and total AMPK antibodies applied on extracts from hearts under depicted conditions and corresponding graphs. Each bar is a mean ± SEM (n = 4–5). AU = arbitrary units.

Fig. 6

Systemic exposure to 18% oxygen phosphorylates S473Akt in the heart. Original Western blots with phospho-S473-Akt, phospho-T308-Akt and total Akt antibodies applied on extracts from hearts under depicted conditions and corresponding graphs. Each bar is a mean ± SEM (n = 4–5). *P < 0.05. AU = arbitrary units.

Fig. 7

LY294002 inhibits phosphorylation of S473Akt, increase of SUR2A in the heart and cardioprotection in response to 18% oxygen. Original Western blots with phospho-S473-Akt, phospho-T308-Akt and total Akt antibodies applied on extracts from hearts under depicted conditions (21% refers to untreated mice exposed to 21% oxygen and 18% to mice treated with LY294002 and exposed to 18% oxygen) and corresponding graphs as well as a graph showing the size of myocardial infarction under depicted conditions. Each bar is a mean ± SEM (n = 3–5). *P < 0.05. AU = arbitrary units.

Fig. 8

Akt regulates LDH/lactate, which, in turn, regulates SUR2A. LY294002 inhibits increase in LDH and lactate in the heart in response to 18% oxygen. Bar graphs depicting LDH (LDH) and lactate (Lactate) levels in extracts from hearts under depicted conditions (21% refers to untreated mice exposed to 21% oxygen and 18% to mice treated with LY294002 and exposed to 18% oxygen). Each bar is a mean ± SEM (n = 4–5).

Fig. 9

LDH activity, but not lactate, seems to regulate SUR2A levels. A–B. Original Western blots with SUR2A antibody applied on extracts from H9c2 cells in the absence (control) and presence of galloflavin (A) or sodium oxamate (B) and corresponding graphs. Each bar is a mean ± SEM (n = 3). C–E. Graphs showing levels of lactate (C), LDH (D) and SUR2A (E) and in untreated cells and cells treated with sodium lactate (30 mM). Each bar is a mean ± SEM (n = 3). F. A graph showing cell survival in H9c2 cells infected by luciferase (control cells labelled as M-LDH) and cell infected by 193gly-M-LDH. Each bar is a mean ± SEM (n = 3–5). *P < 0.05. AU = arbitrary units.