A dual mechanism of cytoprotection afforded by M-LDH in embryonic heart H9C2 cells

Abstract

Muscle form of lactate dehydrogenase (M-LDH), a minor LDH form in cardiomyocytes, physically interacts with ATP-sensitive K+ (K ATP) channel-forming subunits. Here, we have shown that expression of 193gly-M-LDH, an inactive mutant of M-LDH, inhibit regulation of the K ATP channels activity by LDH substrates in embryonic rat heart H9C2 cells. In cells expressing 193gly-M-LDH chemical hypoxia has failed to activate K ATP channels. The similar results were obtained in H9C2 cells expressing Kir6.2AFA, a mutant form of Kir6.2 with largely decreased K+ conductance. Kir6.2AFA has slightly, but significantly, reduced cellular survival under chemical hypoxia while the deleterious effect of 193gly-M-LDH was significantly more pronounced. The levels of total and subsarcolemmal ATP in H9C2 cells were not affected by Kir6.2AFA, but the expression of 193gly-M-LDH led to lower levels of subsarcolemmal ATP during chemical hypoxia. We conclude that M-LDH regulates both the channel activity and the levels of subsarcolemmal ATP and that both mechanism contribute to the M-LDH-mediated cytoprotection.

Fig. 1

Kir6.2AFA and 193gly-M-LDH are efficient dominant negatives of Kir6.2 and M-LDH in H9C2 cells. A. Current–voltage relationships with corresponding original membrane currents in cells filled with pipette solution with (3 mM) and without ATP. Each point represents mean ± SEM (n = 5–6). B. Current–voltage relationships with corresponding original membrane currents in cells filled with ATP-free pipette solution in the absence (control) and presence of glybenclamide (30 μM). Each point represents mean ± SEM (n = 5–6). C. Current–voltage relationships with corresponding original membrane currents in control cells (Kir6.2) and cells infected with Kir6.2AFA filled with ATP-free pipette solution. Each point represents mean ± SEM (n = 5–6). D. Current–voltage relationships with corresponding original membrane currents in control cells (Kir6.2) and cells infected with Kir6.2AFA filled with pipette solution containing ATP (3 mM). Each point represents mean (n = 5–6). E. Current–voltage relationships with corresponding original membrane currents in cells filled with pipette solution containing 3 mM ATP (control) and ATP (3 mM) plus pyruvate (20 mM) plus NADH (20 mM). Each point represents mean ± SEM (n = 5–6). F. Current–voltage relationships with corresponding original membrane currents in control cells (M-LDH) and cells infected with 193gly-M-LDH filled with pipette solution containing ATP (3 mM) plus pyruvate (20 mM) plus NADH (20 mM). Each point represents mean ± SEM (n = 5–6). G. LDH activity in control cells and cells infected with 193gly-M-LDH. Each bar represents mean ± SEM (n = 4). H. LDH assay with anti-Kir6.2 immunoprecipitate of membrane fraction of control cells (control) and cells infected with 193gly-M-LDH.

Fig. 2

Intact Kir6.2 and M-LDH are required for the opening of K_ATP channels induced by chemical hypoxia. (A–C) Current–voltage relationships with corresponding original membrane currents in control cells (A), cells infected with Kir6.2AFA (B) and cells infected with 193gly-M-LDH (C) under control conditions and when exposed to DNP (10 mM). Each point represents mean ± SEM (n = 5). (D) Membrane current at 80 mV under conditions in A–C. Each bar represents mean ± SEM (n = 5).

Fig. 3

Catalytic activity of M-LDH is crucial for cell survival in chemical hypoxia. A bar graph showing a percentage of control cells, and cells infected with Kir6.2 AFA and 193gly-M-LDH that survived treatment with DNP (10 mM). Each bar represents mean ± SEM (n = 6–11). ⁎P < 0.05 when compared to the control and ⁺P < 0.05 when Kir6.2AFA were compared with 193gly-M-LDH.

Fig. 4

M-LDH catalytic activity, but not Kir6.2 K⁺ conductance, is required for counteracting chemical hypoxia-induced decrease in subsarcolemmal ATP. Bar graphs showing luciferase (graphs on the left) and annexin-luciferase (graphs on the right) luminescence in cells infected with Kir6.2AFA under control conditions (normoxia) and after treatment with 10 mM DNP (chemical hypoxia), and in cells infected with 193gly-M-LDH under control conditions (normoxia) and after treatment with 10 mM DNP (chemical hypoxia). Each bar represents mean ± SEM (n = 5). ⁎P < 0.05.