M-LDH physically associated with sarcolemmal K ATP channels mediates cytoprotection in heart embryonic H9C2 cells

Abstract

Muscle form of lactate dehydrogenase (M-LDH) physically associate with K(ATP) channel subunits, Kir6.2 and SUR2A, and is an integral part of the ATP-sensitive K(+) (K(ATP)) channel protein complex in the heart. Here, we have shown that concomitant introduction of viral constructs containing truncated and mutated forms of M-LDH (Delta M-LDH) and 193gly-M-LDH respectively, generate a phenotype of rat heart embryonic H9C2 cells that do not contain functional M-LDH as a part of the K(ATP) channel protein complex. The K(+) current was increased in wild type cells, but not in cells expressing Delta M-LDH/193gly-M-LDH, when they were exposed to chemical hypoxia induced by 2,4 dinitrophenol (DNP; 10mM). At the same time, the outcome of chemical hypoxia was much worse in Delta M-LDH/193gly-M-LDH phenotype than in the control one, and that was associated with increased loss of intracellular ATP in cells infected with Delta M-LDH/193gly-M-LDH. On the other hand, cells expressing Kir6.2AFA, a Kir6.2 mutant that abolishes K(ATP) channel conductance without affecting intracellular ATP levels, survived chemical hypoxia much better than cells expressing Delta M-LDH/193gly-M-LDH. Based on the obtained results, we conclude that M-LDH physically associated with Kir6.2/SUR2A regulates the activity of sarcolemmal K(ATP) channels as well as an intracellular ATP production during metabolic stress, both of which are important for cell survival.

Fig. 1

Infection of H9C2 cells with gly193M-LDH/ΔM-LDH has a dominant-negative effect on M-LDH physically associated with K_ATP channel subunits. (A) Original Western blot of anti-Kir6.2 precipitate of H9C2 control cells and cells infected with gly193M-LDH/ΔM-LDH (the size of M-LDH is 36 kDa; similar results were obtained in three blots). (B) LDH assay with anti-Kir6.2 immunoprecipitate of membrane fraction of control cells (control) and cells infected with gly193M-LDH/ΔM-LDH (similar results were obtained in three experiments). (C) Current–voltage relationships in control cells and cells infected with gly193M-LDH/ΔM-LDH filled with pipette solution containing ATP (3 mM) plus pyruvate (20 mM) plus NADH (20 mM). Each point represent mean (n = 5–6).

Fig. 2

M-LDH physically associated with Kir6.2/SUR2A is required for the K_ATP channels activation in chemical hypoxia. (A) Current–voltage relationships with corresponding original membrane currents in control cells and cells infected with gly193M-LDH/ΔM-LDH when exposed to DNP (10 mM). Each point represents mean ± SEM (n = 5). (B) Membrane current at 80 mV under control conditions (normoxia) and when exposed to DNP (10 mM) in wild type cells and cells infected with gly193M-LDH/ΔM-LDH. Each bar represents mean ± SEM (n = 5).

Fig. 3

M-LDH physically associated with Kir6.2/SUR2A is crucial for cell survival in chemical hypoxia. Bar graph showing a percentage of control cells and cells infected with gly193M-LDH/ΔM-LDH that survived treatment with DNP (10 mM). Each bar represents mean ± SEM (n = 6–11). ^*P < 0.05.

Fig. 4

M-LDH physically associated with Kir6.2/SUR2A is required for counteracting chemical hypoxia-induced decrease in intracellular ATP. Bar graphs showing luciferase luminescence in control cells and cells infected with gly193M-LDH/ΔM-LDH under control conditions (normoxia) and after treatment with 10 mM DNP (hypoxia). Each bar represents mean ± SEM (n = 5). ^*P < 0.05.

Fig. 5

A comparison of susceptibility to chemical hypoxia in cells infected with Kir6.2AFA and gly193M-LDH/ΔM-LDH. Bar graph showing survival of cells infected with Kir6.2AFA and gly193M-LDH/ΔM-LDH when exposed to DNP (10 mM). Each bar represents mean ± SEM (n = 6–8). ^*P < 0.05.