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

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.

Introduction

The muscle form of lactate dehydrogenase (M-LDH) is a minor form of LDH in cardiomyocytes that physically associate with sarcolemmal ATP-sensitive K+ (KATP) channel subunits and is a part of sarcolemmal KATP channel protein complex [1]. KATP channels are gated by intracellular ATP and are viewed as a link between cellular metabolism and membrane excitability. In many different tissues, most notably in the heart, the activation of these channels has been shown to protect the cells against different kinds of metabolic stresses. The mechanism of KATP channels-mediated cardioprotection is still to be fully understood. Traditionally, it has been suggested that the opening of sarcolemmal KATP channels shortens action membrane potential, prevent influx of Ca2+ and intracellular Ca2+ overload, which is the main cause of cellular death in metabolic stress [reviewed in 2]. However, this hypothesis cannot explain how then sarcolemmal KATP channels are protective in cells that do not generate action membrane potential and how it comes that the changes in number of KATP channels have more influence on the heart susceptibility to stress then the changes in the channel activity [2,3]. The answers to these questions might lie in the structure of sarcolemmal KATP channel protein complex. Structurally, the cardiac subtype of KATP channels are heteromultimers composed of Kir6.2 subunit, an inwardly-rectifying K+ channel core primarily responsible for K+ permeance, and SUR2A, a regulatory subunit implicated in ligand-dependent regulation of the channel gating [4]. More recent studies have been suggested that the sarcolemmal KATP channel protein complex may physically associate in vivo with enzymes regulating intracellular ATP levels and glycolysis; one of those has been reported to be M-LDH [5-9]. The functional significance of M-LDH in cardiac myocytes is still unknown.

H9C2 cells are embryonic rat hart myocytes that have been used with success to study cardiac sarcolemmal KATP channels [10,11]. Taking this into consideration, we have taken advantage of these cells to study the function of M-LDH in cardiomyocytes. We report not only that M-LDH-mediated regulation of sarcolemmal KATP channels activity contribute to cell survival during metabolic stress, but that ATP produced by M-LDH mediate cytoprotection independently from the channel activity.

Methods

Heart H9C2 cells and viral constructs

Rat embryonic heart H9c2 cells (ECACC, Salisbury, UK) were cultured in a tissue flask (at 5% CO2) containing Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum and 2 mM glutamine. For electrophysiological experiments, the cells were plated on a 35 × 10-mm culture dish containing 25-mm glass cover-slips. The cells were cultured in incubators (Galaxy, oxygen control model, RS Biotech, Irvine, UK). For the experiments H9C2 cells were infected with adenoviruses construct containing either green fluorescent protein (GFP, gift from C. Sunderland, University of Dundee; cells infected with GFP have served as control cells in this study), Kir6.2AFA (a mutant form of Kir6.2 where the pore GFG was mutated into AFA leading to largely reduced K+ conductance, [12]), gly193-M-LDH (a catalytically inactive mutant of M-LDH, [1]), luciferase and annexin VI-luciferase. All adenoviruses were generated using the AdEasy XL Adenoviral Vector System (Stratagene) as described by the manufacturer. All of the genes were subcloned by PCR using primers containing restrict enzyme sites. gly193-M-LDH was already generated in our laboratory [1] and we have subcloned this gene using the following primers: sense, 5′-GGCAGATCTATGGCAACCCTCAAGGACCA-3′, antisense, 5′-GCCTCGAGTTAGAACTGCAGCTCCTTCT-3′ using gly193-M-LDH as a template. To generate Kir6.2AFA, Kir6.2 gene was subcloned using sense, 5′-GCAGGATCCACCATGCTGTCCCGAAAGGGC-3′, antisense, 5′-GCATCTAG ATCAGGACAAGGAATCTGGAG-3′ and the QuickChange Site-directed mutagenesis kit (Stratagene) was used to generate Kir6.2AFA according to the manufacturer's instructions; the mutagenic primers had the following sequences, sense, 5′-TCCAGGTGACCATTGCATTCGCAGGGCGCATGGTGACA-3′, antisense, 5′-TGTCACCATGCGCCCTGCGAATGCAATGGTCACCTGGA-3′. Luciferase gene was subcloned using the following primers: sense, 5′-GCCTCGAGGCCACCATGGAAGACGCCAAA-3′, antisense, 5′-GCGTAAGCTTACACGGCGATCTTTCCGCC-3′ using pGL3-Enhancer vector (Promega) as a template. For Annexin VI the following primers were used: sense, 5′-GCGGATCCATGGCCAAAATAGCACAGG-3′, antisense, 5′-CGCCTCGA GTCCGTCCTCTCCGCCACACAGAGC-3′ using mouse heart QUICK-Clone™ cDNA (BD Biosciences) as a template. PCR was performed by using the highest fidelity PfuUltra™ DNA polymerase (Stratagene) under the following condition: the PCR was run with a hot start for 2 min at 95 °C, followed by 25 cycles of 0.5 min at 95 °C, 0.5 min at 56 °C, and 1 min at 72 °C; and a final extension 10 min at 72 °C. The PCR products were cloned between the Bgl II and Xho I sites of the pShuttle-CMV vector. All of the positive clones containing DNA inserts were verified by DNA sequencing. After construction, the shuttle vectors were linearized with Pme I and transformed into BJ5183-AD-1 competent cells to perform homologous recombination in Escherichia coli with these shuttle vectors and a large adenovirus-containing plasmid following electroporation. Recombinants were identified from single colonies, linearised, and then transfected into HEK293 cells to produce infective adenovirus virions. Adenoviral particles were obtained by cell extraction after 7–10 days of transfection, and the primary virus was further amplified by infection of AD-293 cultures. The virus titer was determined using QuickTiter Adenovirus Titer Immunoassay Kit (Cell Biolabs, Inc) according to manufacturer's instructions. Typical virus titers were in the 109–1010 pfu/ml range. To infect H9C2 cells, a solution of recombinant adenovirus was mixed with culture medium, and cells were exposed to the virus with a multiplicity of 10 viral particles/cell for 48 h.

Real time RT-PCR

Total RNA was extracted from heart of rat and H9C2 cells using TRIZOL reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's recommendations. Extracted RNA was further purified by RNeasy Plus Mini Kit (Qiagen, Crawley, UK) according to the manufacturer's instruction. The specific primers for rat M-LDH were designed using Beacon Designer 3.0 software (Bio-Rad) as the following, sense, 5′-ACAGAGTTATTGGAAGTGGTTG-3′, antisense, 5′-GGACGCTGAGGAAGACATC-3′, 408 bp of PCR product. The specificity of primers was tested by melting curve analysis and for their ability to produce no signal in negative controls by dimer formation. The RT reaction was carried out with ImProm-II Reverse Transcriptase (Promega, Madison, WI). A final volume of 20 μl of RT reaction containing 4 μl of 5× buffer, 3 mM MgCl2, 20 U of RNasin® Ribonuclease inhibitor, 1 U of ImProm-II reverse transcriptase, 0.5 mM each of dATP, dCTP, dGTP, and dTTP, 0.5 μg of oligo(dT), and 1 μg of RNA was incubated at 42 °C for 1 h and then inactivated at 70 °C for 15 min. The produced cDNA were used as template for the quantitative real-time PCR. A SYBR Green I system was utilized in the reaction. The 25 μl reaction mixture contained: 12.5 μl iQ™ SYBR® Green Supermix (2×), 7.5 nM each primers, 9 μl of ddH2O, and 2 μl of cDNA. The thermal cycling conditions were as follows: an initial denaturation at 95 °C for 3 min, followed by 38 cycles of 10 s of denaturing at 95 °C, 15 s of annealing at 56 °C, and 50 s of extension at 72 °C. The real-time PCR was performed in the same wells of a 96-well plate in the iCycler iQ™ Multicolor Real-Time Detection System (Bio-Rad, Hercules, CA). The data were collected following each cycle and displayed graphically (iCycler iQ™ Real-time Detection System Software, version 3.0A, Bio-Rad, Hercules, CA). Threshold cycle (CT) values were determined automatically by software. The melting curve data were collected to check the PCR specificity. The cDNA sample was always run in duplicate and the corresponding no-RT mRNA sample was included as a negative control [13].

Patch clamp electrophysiology

To monitor whole-cell K+ current the gigaohm seal patch-clamp technique was applied in the whole-cell configuration. For whole-cell electrophysiology applied on H9C2 cells were superfused with Tyrode solution (in mM: 136.5 NaCl; 5.4 KCl; 1.8 CaCl2; 0.53 MgCl2; 5.5 glucose; 5.5 HEPES–NaOH; pH 7.4). All pipettes (resistance 3–5 MΩ), were filled with (in mM): KCl 140, MgCl2 1, EGTA–KOH 5, HEPES–KOH 5 (pH 7.3). Depending whether open or closed KATP channels were required, 3 μM (for ATP-free pipette solution; this small amount of ATP was added to prevent the channel run-down) or 3 mM (to keep KATP channels closed of ATP) of ATP was added. When the effect of LDH substrates on whole-cell K+ current was assessed NADH plus pyruvate (20 mM each) was added into the pipette solution. The effect of 2,4-dinitrophenol (DNP; 10 mM) on K+ current in H9C2 cells was measured using perforated patch-clamp electrophysiology with essentially the same pipette solution as above just ATP was omitted and amphotericin B (Sigma, 240 μg/ml) added. For all cells monitored, the membrane potential was normally held at − 40 mV and the currents evoked by a series of 400 ms depolarising and hyperpolarising current steps (− 100 mV to + 80 mV in 20 mV steps) recorded directly to hard disk using an Axopatch-200B amplifier, Digidata-1321 interface and pClamp8 software (Axon Instruments, Inc., Forster City, CA). The capacitance compensation was adjusted to null the additional whole-cell capacitative current. The slow capacitance component measured by this procedure was used as an approximation of the cell surface area and there was no difference in cell surface area in relation to viral infection irrespective of the gene that was introduced (in all cases the average membrane capacitance was ∼ 250 pF and the difference between control cells and any group of infected cells was less then 7%). Currents were low pass filtered at 2 kHz and sampled at 100 μs intervals.

Cell survival assay

The survival of H9C2 cells were assayed using Multitox-Fluor Multiplex Cytotoxicity Assay (Promega). Briefly, H9C2 cells were plated in complete media (DMEM containing 10% FCS) in 96-well plate, the recombinant adenovirus (GFP, Kir6.2AFA or gly193-M-LDH) was added to the wells at the multiplicity of infection of 10. After 48 h infection, the DNP was added to each well at the final concentration of 10 mM. To measure cell survival 6 h later, the peptide substrate (GF-AFC) that can be cleaved only by live cells was added to the each well. Following 30 min-long incubation at 37 °C, plates were measured using 1420 Multilabel Counter (Victor) plate reader, with excitation at 370 nm and emissions of 480 nm. The percentage of live cells was calculated based on the intensity of fluorescence according to the manufacturer instructions.

Luciferase assay

H9C2 cells were infected with GFP/luciferase (control for total ATP) and GFP/annexin-luciferase (control for subsarcolemmal ATP) or with ΔM-LDH/luciferase (to measure total ATP) and ΔM-LDH/annexin-luciferase (to measure subsarcolemmal ATP) 48 h before luciferase assay. To measure luciferase luminescence cells were mounted in 96-well plate in buffer with the following composition (in mM): 30 HEPES, 3 ATP, 15 MgSO4, 10 DTT; pH: 7.4. Some of the cells were untreated while the others were treated with 10 mM DNP. The reaction for luciferase luminescence measurement was initiated by adding 100 μM of luciferin and the luminescence was measured on a plate reader 1420 Multibabel Counter (Victor). Luminescence was measured in the absence of DNP and after 1 h of cell incubation with 10 mM DNP.

Immunoprecipitation and lactate dehydrogenase assays

Total lactate dehydrogenase (LDH) activity was measured in H9C2 cell extracts. In brief, the cells were homogenised in buffer (TRIS 10 mM, NaH2PO4 20 mM, EDTA 1 mM, PMSF 0.1 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml, at pH = 7.8) and centrifugated at 500 g (to remove large particles from the homogenate), the supernatant was kept at 4 °C for 18 h and the LDH activity was measured using Roche/Hitachi MODULAR analyzer P1800 and Cobas Roche/Hitachi kit according to the manufacturer instructions. To obtain cellular membrane fraction cells were homogenised in buffer I (TRIS 10 mM, NaH2PO4 20 mM, EDTA 1 mM, PMSF 0.1 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml, at pH = 7.8) and incubated for 20 min (at 4 °C). The osmolarity was restored with KCl, NaCl and sucrose and the obtained mixture was centrifugated at 500 g. The supernatant was diluted in buffer II (imidazole 30 mM, KCl 120 mM, NaCl 30 mM, NaH2PO4 20 mM, sucrose 250 mM, pepstatin 10 μg/ml, leupeptin 10 μg/ml, at pH = 6.8) and centrifugated at 7000 g, pellet removed and supernatant centrifugated at 30 000 g. The obtained pellet contains membrane fraction. Lactate dehydrogenase activity was determined in a reagent solution containing 0.2 M TRIS–HCl (100 ml), 6.6 M NADH (1 ml), 30 mM sodium pyruvate (10 ml); pH was adjusted to 7.3 at 25 °C. LDH activity was measured using a spectrophotometer (WPA lightwave, Jencons) set at wavelength 340 nm on pellets (20 μl) dissolved in PBS (total volume was 100 μl) and put in reagent solution (TRIS–HCl 1.4 ml, NADH 50 μl, sodium pyruvate 50 μl). Following 5 min incubation of the reagent solution in the spectrophotometer pellets were added and the absorbance at 340 nm was measured every min until steady-state is reached. The reaction velocity was determined by a decrease in absorbance at 340 nm, resulting from the oxidation of NADH indicative of LDH activity.

Statistical analysis

Data are presented as mean ± S.E.M, with n representing the number of independent experiments. Mean values were compared by the ANOVA followed by Student's t-test or by Student's t-test alone where appropriate using SigmaStat program (Jandel Scientific, Chicago, Illinois). P < 0.05 was considered statistically significant.

Results

The effect of Kir6.2AFA and 193gly-M-LDH on current flowing through KATP channels

Sarcolemmal KATP channels have a similar composition in the cardiac and skeletal muscle [14]. Although H9C2 cells are known to be a cellular phenotype that is in between skeletal muscle and heart muscle cells [15], the findings obtained with them on sarcolemmal KATP channels and cytoprotection has been shown to be relevant for KATP channels in the heart and cardioprotection [10,11]. This is probably not surprising when considering a similarity in KATP channels structure/physiology between heart and skeletal muscle cells. Real time RT-PCR has revealed that there is a slightly higher expression of M-LDH in H9C2 cells then in adult cardiomyocytes, but no statistical significant difference was found (threshold cycle was 14.7 ± 0.1 in H9C2 cells and 15.4 ± 0.3 in adult heart tissue, P = 0.11, n = 4). It is predominant view that KATP channels, normally closed by high intracellular ATP, have to be activated in order to protect cells against metabolic stress (reviewed in [2]). Indeed, removal of ATP from the pipette solution (3 mM was standard concentration of ATP in the pipette solution) has significantly increased whole-cell K+ current (Fig. 1A; in the presence of ATP current at 80 mV was 821 ± 91 pA while in the absence of ATP this value was 1510 ± 264 pA, n = 5–6, P < 0.01). That this current was flowing through KATP channels was further confirmed by the finding that an inhibitor of KATP channels, glybenclamide (30 μM), has blocked the K+ current activated by removal of intracellular ATP (Fig. 1B; the current at 80 mV was 1602 ± 234 in the absence and 814 ± 114 pA in the presence of glybenclamide, n = 5–6, P < 0.01). To study the role that the channel activity plays in cellular responsiveness to stress we have generated adenoviral construct containing a mutant form of the pore-forming Kir6.2 subunit, named Kir6.2AFA. Kir6.2AFA associates with SUR2A and other KATP channel-forming proteins to form a KATP channel protein complex but has vastly decreased ability to conduct K+ current [12]. When we have expressed Kir6.2AFA in H9C2 cells, removal of ATP did not increase K+ current as it happened in cells expressing wild type Kir6.2 (Fig. 1C; whole-cell current at 80 mV in virtually ATP-free pipette solution was 1510 ± 264 pA and 1019 ± 108 pA for cells expressing Kir6.2 and Kir6.2AFA, respectively, n = 5–6, P < 0.01). These findings confirm that KATP channels containing Kir6.2AFA as a pore subunit have largely impaired conductance of K+ ions. That this is an effect specific for KATP channels was further supported by our findings that expression of Kir6.2AFA did not affect whole-cell K+ current when ATP (3 mM) were present the pipette solution (Fig. 1D).

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.

Lactate dehydrogenase (LDH) is a tetramer composed of either M (muscle) and/or H (heart) subunits, which may be combined to form five LDH isozymes. In the heart, all LDH isozymes are present; LDH1 (H4) is the predominant form, whereas LDH5 (M4) is present in trace amounts [16]. It has been shown that M-LDH, but not H-LDH, physically associate with Kir6.2 and SUR2A subunits [1]. However, the functional significance of this particular species of M-LDH in myocytes is yet unknown. To address this question we have generated 193gly-M-LDH, a mutant form of M-LDH that is catalytically inactive [1], into the adenovirus and expressed it in H9C2 cells. It is established that a lactate, a product of LDH activity, acts as a KATP channel opener and can activate the channel even in the presence of millimolar ATP [1]. LDH substrates, pyruvate and NADH, are known to open KATP channels in cardiomyocytes expressing wild type of M-LDH and this is due to lactate production in the microenvironment surrounding the channels [1]. Indeed, in cells expressing wild type of M-LDH, the presence of pyruvate (20 mM) and NADH (20 mM) has increased whole-cell K+ current despite intracellular presence of 3 mM ATP (Fig. 1E; the current at 80 mV was 821 ± 91 pA in the absence and 1740 ± 221 pA in the presence of pyruvate and NADH, n = 5–6, P < 0.01). In contrast, pyruvate (20 mM) and NADH (20 mM) were not able to increase whole-cell K+ current in cells expressing 193gly-M-LDH (Fig. 1F; the current at 80 mV was 1740 ± 221 pA in cells expressing wild type of M-LDH and 748 ± 92 pA in cells expressing 193gly-M-LDH, n = 5–6, P < 0.01). Taken all together, the performed experiments suggested that expression of 193gly-M-LDH has generated a sarcolemmal KATP channel protein complex that cannot be activated by substrates of LDH, which would be in agreement with the notion that catalytic activity of M-LDH physically associated with KATP channel subunits regulates the channel behaviour. At the same time, infection of H9C2 cells with 193gly-M-LDH did not affect total LDH activity (Fig. 1G). However, the LDH activity in Kir6.2 immunoprecipitate was significantly decreased in cells infected with 193gly-M-LDH when compared to control cells (Fig. 1H). This is consistent with the findings that M-LDH is a minor form of LDH in the heart [16] and that M-LDH physically associate with KATP channel subunits to regulate KATP channels activity by regulating concentration of lactate in microenvironment surrounding the channel [1].

Regulation of KATP channels activity during chemical hypoxia by M-LDH

2,4-dinitrophenol (DNP) is known metabolic inhibitor that was used with success to induce metabolic stress in different cell types [17]. When applied, this compound decreases intracellular ATP production and induces chemical hypoxia, which activates KATP channels [18]. In turn, such activation of the channels decreases a degree of cell injury [19]. We have applied perforated patch-clamp electrophysiology to test whether M-LDH activity regulates the opening of KATP channels when cells are challenged with DNP. This method preserves the intracellular milieu during the whole-cell recordings [20], which allowed us to monitor the behaviour of KATP channels-conducted K+ current during stress under conditions of intact intracellular environment. In control H9C2 cells, DNP (10 mM) has induced the activation of KATP channels, as reflected by the increase in whole-cell K+ current (Fig. 2A, D; the current at 80 mV was 610 ± 58 pA under control conditions and 1188 ± 199 pA in the presence of DNP, n = 5, P < 0.01), which was not surprising considering that DNP is known as a metabolic inhibitor that leads to the activation of sarcolemmal KATP channels [18]. Then, we have looked whether we can detect DNP-induced increase in K+ current when cells are infected with Kir6.2AFA mutant. In these cells, DNP (10 mM) did not induce increase in K+ current (Fig. 2B, D; the current at 80 mV was 822 ± 85 pA under control conditions and 534 ± 178 pA in the presence of DNP, n = 5, P = 0.36). It is known that KATP channels containing Kir6.2AFA instead of Kir6.2 have largely decreased K+ conductance and the obtained results are in agreement with the idea that DNP-induced increase in K+ current was blocked due to Kir6.2AFA being a pore-forming subunit of the KATP channel in H9C2 cells. It has been shown previously that M-LDH can regulate KATP channels behaviour in a recombinant heterologues expression system [1]. However, the role of M-LDH in regulating sarcolemmal KATP channels in its native environment during metabolic stress is yet unknown. In cells infected with catalytically inactive form of M-LDH, 193gly-M-LDH, DNP (10 mM) did not crease K+ current (Fig. 2C, D; the current at 80 mV was 618 ± 52 pA under control conditions and 454 ± 185 pA in the presence of DNP, n = 5, P = 0.44) suggesting that M-LDH activity contributes to the activation of KATP channels during metabolic stress. This would confirm hypothesis that M-LDH-produced lactate in microenvironment surrounding the channel is a factor involved in the channel opening in stressful conditions.

Fig. 2

Intact Kir6.2 and M-LDH are required for the opening of KATP 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).

Regulation of cell survival by M-LDH and KATP channels activity during chemical hypoxia

In some studies, a failure to activate KATP channels has been associated with the exacerbation of the outcome of the metabolic stress [19], but in some others that was not the case [21]. Here, we have assessed whether mutations of Kir6.2 and M-LDH would affect in any way the outcome of metabolic stress with DNP. Thus, only 45.1 ± 1.8% (n = 11) of control cells survived exposure to DNP (10 mM) (Fig. 3). When the same type of stress was imposed on cell infected with Kir6.2AFA, survival was just slightly decreased (39.2 ± 0.9%, n = 8, Fig. 3), but the difference was statistically significant (P = 0.02). On the other hand, cells infected with 193gly-M-LDH have been much more susceptible to stress, as only 27.1 ± 4.8% cells have survived 10 mM DNP (Fig. 3, n = 6). The cells expressing 193gly-M-LDH were not only more susceptible to stress when compared to the controls (P < 0.001, Fig. 3), but also when compared to Kir6.2AFA cells (P = 0.01, Fig. 3). Although in both, Kir6.2AFA and 193gly-M-LDH, cellular phenotypes KATP channels were silent during stress (Fig. 2), the outcome of the stress was much worse in cells infected with 193gly-M-LDH than in those infected with Kir6.2AFA. A concomitant infection of cells with Kir6.2AFA and 193gly-M-LDH did not additively decreased the cell survival in the presence of DNP (10 mM; survival of these cells was 35.3 ± 4.1%, n = 3, P = 0.14 when compared to cells infected by 193gly-M-LDH alone).

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.

Regulation of intracellular and subsarcolemmal ATP levels by M-LDH and KATP channels activity

The results obtained with Kir6.2AFA and 193gly-M-LDH on cell survival has suggested that KATP channel-mediated cytoprotection could be, at least in part, independent of the channel activity. Here, we have elucidated the effect of DNP on total and subsarcolemmal ATP in control cells, cells infected with Kir6.2AFA and cells infected with 193gly-M-LDH. To be able to measure intracellular and subsarcolemmal ATP in living cells, we have infected cells with constructs encoding luciferase gene and annexin-luciferase, which is a modified technique by Kennedy et al. [22]. It has been shown that this strategy allows measurement of ATP levels in cytosol (luciferase) and subsarcolemmal domains (annexin-luciferase). Infection with Kir6.2AFA per se did not affect either total or subsarcolemmal ATP levels (total ATP: the intensity of luminescence was 283.2 ± 9.1 AU in control and 302.3 ± 6.7 AU in Kir6.2AFA cells, n = 5, P = 0.18; subsarcolemmal ATP: the intensity of luminescence was 596.0 ± 59.1 AU in control and 702.0 ± 29.4 AU in Kir6.2AFA cells, n = 5, P = 0.15; Fig. 4). In the presence of DNP, luciferase luminescence was 71.8 ± 2.9 in control cells and 72.8 ± 2.7 in Kir6.2AFA cells (n = 5, P = 0.80; Fig. 4). On the other hand, annexin-luciferase luminescence in the presence of DNP (10 mM) was 110.2 ± 11.3 AU in control and 115.8 ± 21.8 AU in Kir6.2AFA cells (n = 5, P = 0.83, Fig. 4). The infection of cells with 193gly-M-LDH also did not affect total or subsarcolemmal ATP levels under control conditions (total ATP: the intensity of luminescence was 243.0 ± 17.0 AU in control and 215.1.3 ± 11.4 AU in 193gly-M-LDH cells, n = 5, P = 0.19; subsarcolemmal ATP: the intensity of luminescence was 541.6 ± 86 AU in control and 529.4 ± 38.2 AU in 193gly-M-LDH cells, n = 5, P = 0.90; Fig. 4). Treatment of cells with DNP (10 mM) induced similar decrease of total ATP in both control and 193gly-M-LDH cells (61.0 ± 2.2 AU in control and 62.4 ± 2.2 AU in infected cells, n = 5, P = 0.67; Fig. 4). In contrast, the magnitude of DNP-induced decrease of subsarcolemmal ATP was significantly higher in cells infected with 193gly-M-LDH (73.6 ± 5.4 AU, n = 5, Fig. 4) then in control cells (92.0 ± 5.8 AU, n = 5, P = 0.04; Fig. 4).

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.

Discussion

In the present study, we have shown that the catalytic activity of M-LDH is essential for cellular survival during metabolic stress. It seems that this cytoprotective effect of M-LDH is associated with the regulation of sarcolemmal KATP channels activity and subsarcolemmal ATP production.

It has been previously shown that lactate, a catalytic product of LDH, can activate KATP channels despite high intracellular ATP. The present study shows that the catalytic activity of M-LDH is required for the channel opening during metabolic stress. It is well established that 100 μM of ATP is sufficient to completely close sarcolemmal KATP channels and this low concentration is never achieved in vivo, even during most the severe stress [2]. As an example, we have shown in the present study that a very high concentration of metabolic inhibitor, DNP, has decreased luciferase luminescence, the measure of total ATP, for ∼ 4 times. Taking into consideration that the physiological concentration of intracellular ATP is 6–10 mM it would mean that under DNP the level of ATP was ∼ 2 mM, which is a concentration of ATP more then sufficient to keep the channels closed. The obtained results support the notion that the level of ATP is not a sole factor regulating the activity of KATP channels in vivo and suggest a role for M-LDH as a lactate producer, a compound that can open the channels in the presence of millimolar ATP [1].

The mechanism of cytoprotection afforded by KATP channels is a long standing issue. The structural studies of these channels have shown that channel subunits in vivo are physically associated with enzymes that catalyses ATP production [5-9], including M-LDH, which activity is particularly important under anaerobic conditions (reviewed in 16). Such composition of sarcolemmal KATP channels would allow a supposition that KATP channel could serve as ATP-producing machinery. The fact that Kir6.2AFA did not affect total ATP and subsarcolemmal ATP levels would stand against a possibility that the changes in the channel activity alone affect the ATP production either in subsarcolemma or anywhere else in the cell. It seems that the channel activity per se have no bearing on subsarcolemmal and intracellular ATP production. In contrast, although a loss of catalytic activity of M-LDH did not affect total intracellular ATP levels during stress, it did facilitate ATP loss in subsarcolemmal domain. This suggests that M-LDH is an ATP producer important for maintaining subsarcolemmal ATP during metabolic stress. M-LDH seem to be is essential for 1) the activation of sarcolemmal KATP channels and 2) for maintaining subsarcolemmal levels of ATP during stress. The fact that the inhibition of M-LDH catalytic activity had significantly worse outcome on cell survival then those with the inhibition of the KATP channels conductance alone suggests that the level of subsarcolemmal ATP is a relevant factor in defining the outcome of the metabolic stress. As M-LDH is an integral part of the sarcolemmal KATP channel protein complex, this implies that the KATP channels-mediated cytoprotection involves not only regulation of membrane potential [23,24], but also a regulation of subsarcolemmal levels of ATP. The obtained findings would support the idea that KATP channels could have a role in regulating cardiac bioenergetics that is yet beyond the channel activity. At the same time, it seems that M-LDH is an important element in cytoprotection afforded by KATP channels. A unique property of M-LDH is that it produces a lactate that can open the channel despite millimolar presence of ATP and, at the same time, it produces ATP, which provide double benefits for cells exposed to metabolic stress: 1) hyperpolarisation of membrane and decrease of Ca2+ influx/Ca2+ loading and 2) production of ATP in subsarcolemmal regions and counteracting stress-induced impairments of energy-dependent membrane transport processes.

On first sight, an increase in subsarcolemmal ATP and KATP channels activation might look as being in contradiction. However, it is known for years that ischemia opens sarcolemmal KATP channels before the fall of intracellular ATP occur, suggesting that the intracellular level of ATP is not the only factor that regulates the channel activity [25]. This is supported by numerous evidences. As an example, in excised membrane patches, 100 μM of ATP is sufficient to abolish sarcolemmal KATP channels opening while in vivo KATP channels are activated by hypoxia/ischaemia despite intracellular ATP being within millimolar range [26]. Today, it is recognised that the activity of KATP channels is controlled by a complex interaction of many intracellular factors and signalling pathways. In addition to ATP, it has been suggested that the activity of these channels may be regulated by other nucleotides [27,28], intracellular pH ([29,30], lactate [1,31], cytoskeleton [32,33], protein kinase C [34], phosphatidylinositol-4,5-bisphosphate [35], and by the operative condition of the channel itself [36,37]. It is well established that lactate activates KATP channels in the presence of millimolar ATP, though the mechanism of this activation is yet unknown [1,31]. It has been shown that intracellular acidification decreases sensitivity of KATP channel subunits to ATP, but there is no evidence that this is associated with the effect of lactate. It has been previously shown that treatment with DNP decreases pH in cardiac cells [38] and this acidification would certainly activate sarcolemmal KATP channels. We have shown in this study that the catalytic activity of M-LDH is required for the channels activation, which is presumably mediated by lactate. In addition (or in connection) with lactate could be also acidification of the subsarcolemmal regions that would contribute to the KATP channels opening and, thereby, to cell survival. It has been previously shown, at both native and recombinant levels, that the activation of KATP channels prevents DNP-induced membrane depolarisation [23,24], which seems to decrease influx of Ca2+ by inhibiting the activation of voltage gated Ca2+ channels and preventing intracellular Ca2+ overload [23,39-41]. Thus, the activation of KATP channels is crucial for cytoprotection, but it also seems that a little bit of ATP produced by M-LDH also helps. As cells expressing Kir6.2AFA (inhibits KATP channels activity without affect on subsarcolemmal ATP) were more resistant then the cells expressing 193glyM-LDH (inhibits both the KATP channels activity and slightly decrease subsarcolemmal ATP in the presence of ATP) it seems that the M-LDH effect on subsarcolemmal ATP levels contribute to cytoprotection afforded by M-LDH. The fact that co-infection with Kir6.2AFA/193glyM-LDH did not further decrease cell survival when compared to 193glyM-LDH alone confirm conclusion that Kir6.2AFA and 193glyM-LDH share the same mechanism of increase in cellular susceptibility (which is an inhibition of the channel activity) with 193glyM-LDH being more deleterious due to the effect on subsarcolemmal ATP production that Kir6.2AFA does not have.

Taken all together this study has demonstrated that M-LDH seems to be crucial in regulating KATP channels activity, ATP production and cellular resistance to metabolic stress. Consequently, it seems that cytoprotection afforded by KATP channels is mediated not only by the channel activity, but also by the production of ATP.