Infection with AV-SUR2A protects H9C2 cells against metabolic stress: a mechanism of SUR2A-mediated cytoprotection independent from the K(ATP) channel activity

Abstract

Transgenic mice overexpressing SUR2A, a subunit of ATP-sensitive K(+) (K(ATP)) channels, acquire resistance to myocardial ischaemia. However, the mechanism of SUR2A-mediated cytoprotection is yet to be fully understood. Adenoviral SUR2A construct (AV-SUR2A) increased SUR2A expression, number of K(ATP) channels and subsarcolemmal ATP in glycolysis-sensitive manner in H9C2 cells. It also increased K(+) current in response to chemical hypoxia, partially preserved subsarcolemmal ATP and increased cell survival. Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K(+) conductance, abolished the effect of SUR2A on K(+) current, did not affect SUR2A-induced increase in subsarcolemmal ATP and partially inhibited SUR2A-mediated cytoprotection. Infection with 193gly-M-LDH, an inactive mutant of muscle lactate dehydrogenase, abolished the effect of SUR2A on K(+) current, subsarcolemmal ATP and cell survival; the effect of 193gly-M-LDH on cell survival was significantly more pronounced than those of Kir6.2AFA. We conclude that AV-SUR2A increases resistance to metabolic stress in H9C2 cells by increasing the number of sarcolemmal K(ATP) channels and subsarcolemmal ATP.

Fig. 1

Infection with AV-SUR2A increases expression of SUR2A without affecting the expression of other K_ATP channel subunits. Bar graphs represent cycling thresholds of the real time RT-PCR progress curves of K_ATP channel-forming subunits. Inset in SUR2A graph is a Western blot of anti-Kir6.2 immunoprecipitate from H9C2 cells with either anti-SUR2 or anti-GAPDH antibody under depicted conditions (for SUR2 the signal depicted was between 97 and 191 kDa while for GAPDH it was ∼ 39 kDa as determined by SeeBlue Plus Prestained Standard, Invitrogen; similar results were obtained in three independent experiments). Each bar represents mean ± SEM (n = 4 for each). *P < 0.05.

Fig. 2

Infection with AV-SUR2A increases DNP-induced whole cell K⁺ current. Original membrane currents under depicted conditions (DNP concentration was 10 mM) and line-scatter graph showing DNP-induced K⁺ current component (obtained by substraction of whole cell K⁺ current in the presence and absence of DNP) in control cells (control) and cells infected with AV-SUR2A (SUR2A). Each point represents mean ± SEM (n = 5 for each).

Fig. 3

Infection with AV-SUR2A increases survival of cells exposed to DNP. A bar graph showing a percentage of survival in control cells and cells infected with SUR2A alone, Kir6.2 alone or SUR2A/Kir6.2 exposed to DNP (10 mM). Each bar represent mean ± SEM (n = 3–14). *P < 0.05 when compared to control.

Fig. 4

Kir6.2AFA abolishes SUR2A-mediated increase in K⁺ current. Original membrane currents under depicted conditions (DNP concentration was 10 mM) and line-scatter graph showing DNP-induced K⁺ current component (obtained by substraction of whole cell K⁺ current in the presence and absence of DNP) in cells infected with AV-SUR2A (SUR2A) and AV-SUR2A plus AV-Kir6.2AFA. Each point represents mean ± SEM (n = 4 for each).

Fig. 5

Kir6.2AFA only partially inhibits SUR2A-mediated cytoprotection. A bar graph showing a percentage of survival in cells infected with Kir6.2AFA alone, Kir6.2AFA/SUR2A and SUR2A alone exposed to DNP (10 mM). Each bar represent mean ± SEM (n = 5–14). *P < 0.05 when compared to the control.

Fig. 6

Infection with AV-SUR2A increases the levels of subsarcolemmal ATP. Bar graphs showing luciferase (intracellular ATP) and annexin-luciferase (subsarcolemmal ATP) luminescence in control cells and cells infected with AV-SUR2A under control conditions (control) and after treatment with either 10 mM DNP (DNP) or 50 mM 2-deoxyglucose (2-deoxyglucose). Each bar represent mean ± SEM (n = 5). *P < 0.05.

Fig. 7

Kir6.2AFA does not modify the effect of AV-SUR2A on ATP levels. Bar graphs showing luciferase (intracellular ATP) and annexin-luciferase (subsarcolemmal ATP) luminescence in cells infected with kir6.2AFA alone and SUR2A/Kir6.2AFA under control conditions (control) and after treatment with 10 mM DNP (DNP). Each bar represent mean ± SEM (n = 5). *P < 0.05.

Fig. 8

193gly-M-LDH inhibits effects of SUR2A on DNP-induced K⁺ current and ATP levels. A. Line-scatter graph showing DNP-induced K⁺ current component in cells infected with AV-193gly-M-LDH in the absence (control) and presence of DNP (10 mM). Each point represents mean (n = 4). B. Bar graphs showing luciferase (intracellular ATP) and annexin-luciferase (subsarcolemmal ATP) luminescence in cells infected with SUR2A alone and SUR2A/193gly-M-LDH under control conditions (control) and after treatment with 10 mM DNP (DNP). Each bar represent mean ± SEM (n = 5). *P < 0.05.

Fig. 9

Infection with AV-SUR2A increases survival of cells exposed to DNP. A bar graph showing a percentage of survival in cells infected with SUR2A alone, SUR2A/Kir6.2AFA and SUR2A/193gly-M-LDH exposed to DNP (10 mM). Each bar represent mean ± SEM (n = 3–6). *P < 0.05 when compared to SUR2A and ⁺P < 0.05 when compared to SUR2A/Kir6.2AFA.