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FEBS Lett. Author manuscript; available in PMC 2011 January 4.
Published in final edited form as:
PMCID: PMC2794998
NIHMSID: NIHMS158923

Cardioprotective Activity of A Novel and Potent Competitive Inhibitor of Lactate Dehydrogenase

Abstract

Alkaline incubation of NADH results in the formation of a very potent inhibitor of lactate dehydrogenase. High resolution mass spectroscopy along with NMR characterization clearly showed that the inhibitor is derived from attachment of a glycolic acid moiety to the 4-position of the dihydronicotinamide ring of NADH. The very potent inhibitor is competitive with respect to NADH. The inhibitor added in submicromolar concentrations to cardiomyocytes protects them from damage caused by hypoxia/reoxygenation stress. In isolated mouse hearts, addition of the inhibitor results in a substantial reduction of myocardial infarct size caused by global ischemia/reperfusion injury.

Keywords: Lactate Dehydrogenase, Competitive inhibition, Enzyme kinetics, cardioprotection

1. Introduction

For many years analogues of NAD+/NADH have proven valuable in studies of the binding of the pyridine coenzyme and mechanism of dehydrogenases which utilize these cofactors [1]. Recently it was demonstrated that incubation of NADH in oxygen saturated alkaline solutions results in the formation of a very strong competitive inhibitor of mitochondrial complex I (NADH:ubiquinone oxidoreductase) [2]. Many NAD+/NADH derivatives may act as alternative substrates or competitive inhibitors of lactate dehydrogenase (LDH). They originate from modification of the nicotinamide ring of the dinucleotide at positions C-3, C-4, or C-5 [1, 3-5]. These compounds were shown to act as competitive inhibitors with respect to NADH and were capable of inhibiting both LDH and malate dehydrogenase (MDH). Other dehydrogenases such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glycerate dehydrogenase, and glycerol dehydrogenase were only slightly suppressed by the NADH derivatives [5]. It has been demonstrated that incubation of LDH in the presence of pyruvate and NAD+ results in the formation of a strong inhibitor (NAD-Pyr) in the active center of the enzyme. In NAD-Pyr the β-carbon atom of pyruvate is covalently linked to the C-4 position of the nicotinamide ring [6-8]. The NAD- and 3-substituted NAD (APAD) pyruvate adducts also spontaneously form during the incubation of pyruvate and the dinucleotides at pH 11 [9, 10]. Addition of these adducts to LDH results in strong suppression of enzyme activity [11, 12]. These adducts are, however, relatively unstable at neutral pH and rapidly decompose into pyruvate and NAD or APAD [9, 12].

During ischemia cells produce high intracellular levels of lactic acid. In ischemic tissues glycolysis provides the main source of ATP for the cell. The final product of glycolysis, pyruvate, can not be further metabolized by mitochondria during anaerobiosis and is converted to lactate by LDH. The latter reaction leads to the accumulation of lactic acid and to the associated lowering of intracellular pH. It has been proposed that the decrease in pH caused by lactic acid accumulation may be a major cause of severe ischemic brain and heart injury [13-16]. The key role of LDH in the above process stimulated the development of specific inhibitors of the enzyme, which could prevent the production of lactic acid and thus protect tissues from lactic acidosis. It has been shown that reduced APAD-Pyr strongly decreases lactate production in crude brain extracts [9]. It was, however, much less effective at inhibiting lactate production in synaptosomes. The main reason for a weaker effect on lactate production in vivo is likely associated with the low stability of the inhibitor at physiological pH [12]. This is a serious disadvantage that limits the use of NAD-Pyr and APAD-pyr adducts for preventing lactic acidosis induced damage in vivo.

In this work a novel NADH derived inhibitor of LDH was prepared and its molecular structure characterized. It is shown that the inhibitor is very strong and competitive with respect to NADH. The inhibitor also reduces myocardial infarct size following ischemia/reperfusion injury in mouse heart and protects cultured adult mouse cardiomyocytes from damage induced by hypoxia/reoxygenation. The mechanism of the cardioprotective effect of the LDH inhibitor is discussed.

2. Materials and methods

Preparation of the inhibitor

The crude inhibitor was produced from aerobic alkaline NADH solutions by procedures similar to that that were used to produce a previously described inhibitor of Complex I [2]. Initially 17.5 g of NADH was dissolved in 500 ml of water in a 1 liter capacity plastic bottle with screw top. The bottle was placed on ice and 5 ml of 10 M KOH were added dropwise to the solution with constant stirring at high speed. Then 500 ml of methanol was added and the mixture saturated with oxygen by bubbling the pure gas through the solution for 30 min at 4 °C. The bottle was tightly sealed and left in the dark at room temperature for two days. An intense yellow color developed during the incubation. The solution was diluted twice with double distilled water and applied to a 130 ml capacity Q-sepharose Fast Flow column previously equilibrated with 5 mM K-Pi (pH 12). Elution of the inhibitor was at pH 12 with a linear 5-1000 mM K-Pi gradient at a flow rate of 2 ml/min. The fractions containing the inhibitor eluted from the column after authentic NADH at approximately 400-450 mM K-Pi, (see Supplementary Figure 1). Fractions were tested for their ability to suppress LDH activity. The inhibitor-containing fractions were pooled and concentrated on a C-18 column as follows. The pH of the eluted solution was adjusted to 7.4 by adding 4 M HCl. The neutralized solution (~ 200 ml) was applied to a column pre-packed with 20g of C-18 silica-based matrix column and equilibrated with 0.1 mM K-Pi (pH 7) and eluted from the column in 20% methanol. In order to completely purify the inhibitor we took advantage of the very strong affinity of the inhibitor to LDH. Therefore, the eluate containing crude inhibitor was mixed with an equal volume of a 5 mg/ml LDH solution and incubated at 25 °C for 30 min. The solution was transferred into Centriprep YM-30 centrifugal filter units (Millipore, USA) and LDH in complex with inhibitor was concentrated by centrifugation at 1,800 x g for 30 min at 4 °C. The filtrate was discarded; the resulting concentrate was diluted 10-times by adding 0.1 M K-Pi (pH 7). The solution was then centrifuged a second time. The concentrate obtained after four more separating spins containing a complex of LDH with the inhibitor was collected. The volume of the fraction was adjusted to 10 ml by adding 0.1 M K-Pi (pH 7). 10 ml of 1 M K-Pi (pH 12) was then added to the fraction and incubated for 10 min at room temperature. This treatment leads to denaturation of LDH and to liberation of the free inhibitor from the complex. The solution was transferred into Centriprep YM-30 centrifugal filter units and centrifuged at 1,800 x g for 30 min at 4 °C. The concentrate containing denatured LDH was discarded and the filtrate containing the free inhibitor was collected and used in this study. HPLC analysis of the isolated inhibitor showed that it was chromatographically pure and the final yield was approximately 2 mg of inhibitor from the initial 17.5 g of NADH.

Enzyme assay

Each enzyme was assayed spectrophotometrically at 25 °C in 0.1 M K-Pi (pH 7.0) buffer containing 0.1 mM EDTA. The change of NADH absorbance at 340 nm (ε340=6.22 mM−1 cm−1) was followed. The enzyme assay systems also contain: LDH - 50 μM NADH and 1 mM pyruvate; MDH - 150 μM NADH and 0.25 mM oxaloacetate; alcohol dehydrogenase - 5 mM NAD+ and 3% ethanol; glutamate dehydrogenase – 150 μM NADH, 10 mM α-ketoglutarate and 10 mM ammonium-acetate; DT diaphorase - 100 μM NADH and 60 μM Q1; NADH-oxidase - 100 μM NADH and 0.05 μg/ml of gramicidin D. Other details of the assays are indicated in the figure legends. All reactions were initiated by the addition of enzymes listed below to the assay mixture. LDH (EC 1.1.1.27) from bovine heart and rabbit muscle, MDH (EC 1.1.1.37) from porcine heart, alcohol dehydrogenase (EC 1.1.1.1) from equine liver; glutamate dehydrogenase (EC 1.4.1.3) from bovine liver were all from Sigma (USA). Human recombinant DT diaphorase (EC 1.6.99.2) was prepared as described [17]. Bovine heart submitochondrial particles (SMP) were prepared as described [18].

Absorption Spectroscopy

Absorption measurements were carried out with an Agilent 8453 Diode Array Spectrophotometer (Agilent Technologies, USA).

High Resolution Mass Spectroscopy

The HRMS estimation of the molecular mass and the molecular formula of the inhibitor were done by M-Scan Inc (West Chester, PA, USA) using high resolution ESI-MS in the negative ion mode.

Nuclear Magnetic Resonance Spectroscopy

NMR spectra were determined in D2O (Aldrich, Milwaukee, USA). NMR spectra were recorded with Varian UnityINOVA 700 MHz and 500 MHz spectrometers. TOCSY spectra [19] were recorded with a mixing time of 80 ms. {1H-13C}-HSQC [20] and {1H-13C}-HMBC [21] were optimized for 1JC-H=135 Hz and 2,3JC-H= 10 Hz, respectively. HSQC and TOCSY experiments were recorded using states-TPPI procedure for quadrature detection. In all 2D experiments the time domain data consisted of 2048 complex points in t2 and 400-512 fids in t1 dimension. The relaxation delay was kept at 1.2 s for all experiments. The spectra were acquired at T=5°C and calibrated relative to HDO (4.96 ppm) as internal standard. The NMR data were processed on a SGI Octane workstation using FELIX 98 software (Accelrys, San Diego, USA) and on an iMAC using iNMR (www.inmr.net) software.

In vitro studies in adult mouse cardiac myocytes

Ventricular myocytes were cultured as previously described [22]. On the day after isolation and culture, cardiac myocytes were subjected to 3 hr of hypoxia (1% CO2 and 99% N2). Immediately after hypoxia cells were treated with varying concentrations of NADH-GA ranging from 10 nM to 1 μM during reoxygenation which lasted for 16h. Cardiac myocyte survival was measured as previously described [22] by staining cells in tissue culture dishes with trypan blue solution.

Ischemia/reperfusion protocol

Hearts from male C57/Bl6 mice weighing 20-25 gm were subjected to 40 min of global ischemia and 45 min of reperfusion on a Langendorff apparatus as previously described [23]. Hearts were treated with vehicle (Krebs-Henseleit buffer) or NADH-GA infusion at a concentration of 100 nM beginning at the end of ischemia and lasting throughout reperfusion. Left ventricular pressures were recorded continuously throughout the experiment and infarct size (triphenyltetrazolium method) was measured at the end of reperfusion as previously described [23].

Materials

All fine chemicals used in this study were from Sigma-Aldrich.

3. Results

The exact molecular mass of the purified inhibitor prepared as described in Materials and methods was determined by high resolution electron spray ionization mass spectral (HRMS) analysis in negative ion mode. The purified inhibitor showed a single pseudomolecular ion peak at m/z 738.1182 [(M-H)] (data not shown). This mass corresponds to a molecular formula of C23H30N7O17P2 suggesting the addition of two carbon, three oxygen, and two hydrogen atoms to NADH. In order to define the molecular structure of the purified inhibitor 1D 1H- and 31P-NMR spectra, and 2D COSY, 2D TOCSY, 2D HSQC, and 2D HMBC NMR data were collected. The analysis of the aforementioned experiments allowed us to assign all the 1H, 13C, and 31P resonances of the molecule (Table 1). The NMR results presented in Table 1 along with the HRMS data led us to conclude that the inhibitor originates from the covalent attachment of a glycolic acid (GA) moiety to the C-4 position of the nicotinamide ring (Fig. 1A) and that the inhibitor is essentially pure. In the present study the inhibitor is referred to as NADH-GA.

Figure 1
Molecular structure and absorption properties of NADH-GA
Table 1
1H, 13C, and 31P-NMR assignment of NADH-GA. 1H-13C long range correlation (HMBC) are also reported (700MHz, T=5°C)

The UV/Vis absorption spectrum of NADH-GA is shown in Fig. 1B. The shape of the spectrum resembles that of authentic NADH in the reduced state. NADH has absorption peaks at 260 nm and 340 nm, while the oxidized form (NAD+) only absorbs at 260 nm. The presence of the peak at 327 nm in Fig. 1B is consistent with the NMR data, demonstrating that the nicotinamide residue in NADH-GA is reduced. The inhibitor is stable at neutral pH as incubation of NADH-GA at 4 °C for six days does not result in any noticeable change of the absorption spectrum or the ability of the inhibitor to suppress the activity of LDH. Incubation of the inhibitor at 37 °C resulted in a slow (~half-time of 20 hours) reduction of the absorption at 327 nm and a corresponding decrease in inhibition potency (data not shown).

Addition of 0.2 μ-M NADH-GA in the LDH assay results in a rapid (several seconds) and almost complete suppression of enzyme activity (Fig. 2, trace 1). The NAD+ utilizing enzyme MDH is also partially inhibited by NADH-GA (Fig. 2, trace 2) although 5 times the amount of the inhibitor is required to suppress MDH activity to the same extent as that of LDH (data not shown). Other NAD+/NADH utilizing enzymes were also investigated. It was found that 10 μ-M NADH-GA had no detectable effect on the activity of alcohol dehydrogenase, glutamate dehydrogenase, DT-diaphorase, and NADH oxidase activity catalyzed by bovine heart SMP. These results are consistent with the remarkable similarity in the conserved functional active site amino acids, the nature of the substrates and catalytic mechanism of LDH and MDH [24-26]. The other dehydrogenases tested here are less similar to LDH compared to MDH and would be expected to be less sensitive to an LDH inhibitor.

Figure 2
Kinetics of NADH-GA induced inhibition of LDH and MDH

Preincubation of LDH with NADH-GA prior to initiation of the reaction results in complete inhibition of the initial rate of NADH oxidation followed by a relatively slow activation of the enzyme (Fig. 3A). The slow activation (slow release of the inhibitor) induced by addition of NADH into the assay is consistent with competitive inhibition with very high affinity. The inhibition measured here using kinetic analysis by the Dixon method appears to be in the range of a Ki of ~4 nM, however, this value is most likely overestimated as a result of the tight-binding nature of the inhibitor. Decreasing the incubation temperature from 25° to 10 °C slows down the rate of activation (Fig. 3A, compare traces 1 and 2). Enzyme activation at 10 °C is slow enough to permit accurate estimation of the activation rate and initial rates of the reaction catalyzed by the partially inhibited enzyme using conventional absorption spectroscopy. The first order kinetics of enzyme activation at 10 °C yielded an activation constant (koff) of 1 min−1 (inset to Fig. 3A). Figure 3B presents the dependence of the initial rate of the reaction (10 °C) on the concentration of NADH-GA in the incubation. As seen in Fig. 3B, the initial rate of the reaction, which is proportional to the concentration of free (non-inhibited enzyme), decreases linearly with the amount of inhibitor added. Almost complete inhibition of the initial rate was achieved by addition of ~60 μl of NADH-GA from a stock solution to 1 ml of 0.6 mg/ ml LDH (see Fig. 3B). This end point on the titration curve corresponds to equal concentrations of the inhibitor and the LDH monomer. The concentration of LDH monomers were estimated spectrophotometrically using the molecular extinction of ~50 mM−1 cm−1 at 280 nm [27, 28] and is equal to 19.2 μM. It has been shown that the four coenzyme binding sites of the LDH tetramer are equal and independent [29], suggesting that all four active sites of the enzyme are equivalent with respect to inhibitor binding. The absorption of the inhibitor in the complex at 327 nm is equal to 0.125 corresponding to the extinction coefficient of NADH-GA of 6.5±0.5 mM−1 cm−1. This value is similar to that of NADH and its derivatives in the near-UV spectral region [7, 8, 30].

Figure 3
Inhibition of LDH by NADH-GA

Figure 4A shows the results of treating cultured adult mouse cardiac myocytes with varying concentrations of NADH-GA ranging from 10 nM to 1 μM during 16 hr of reoxygenation following 3 hours of hypoxia. Control cells were incubated under normoxic conditions for the duration of the experiment and are assigned a survival of 100%. Hypoxia/reoxygenation reduced myocyte survival in the untreated control cells to less than 70%, while treatment with NADH-GA improved viability as measured by trypan blue exclusion to 85-90%. These observations demonstrate that as little as 10 nM NADH-GA is sufficient to promote cell survival under conditions of oxidative stress. As noted in the figure legend 1 nM NADHGA or concentrations above 2.5 μM did not result in significantly increased viability of cells following hypoxia/reoxygenation. As shown in Fig. 4B, infusion of NADH-GA at a concentration of 100 nM starting at the end of ischemia and continuing throughout the entire 40 minutes of reperfusion resulted in an almost 3-fold improvement in left ventricular developed pressure at the end of reperfusion. Concurrently infarct size was reduced by an average of 44% compared to vehicle-treated hearts. These data indicate that the inhibitor exerted a substantial prosurvival effect in the ex vivo mouse heart by markedly improving hemodynamic recovery and reducing the extent of tissue damage. These observations are consistent with the prosurvival effects noted in cultured adult mouse cardiac myocytes subjected to oxidative stress (Fig. 4A).

Figure 4
Protective effect of NADH-GA on cardiac myocytes and on mouse heart

4. Discussion

There has been intense interest in the prevention and treatment of acute ischemia/reperfusion injury which results in both short- and long-term damage to heart muscle and which is considered to be the pathophysiology underlying acute myocardial infarction in humans [31]. When coronary blood flow is reduced or ceases, aerobic processing of metabolites via the Krebs cycle is markedly impaired and glycolytic production of pyruvate is rapidly converted to lactate via activation of LDH. Accumulation of lactate during the ischemic phase results in tissue and cellular acidosis [31]. Thus, it was of interest to test the efficacy of a potent stable LDH inhibitor in promoting survival in cultured cardiac myocytes and reducing myocardial infarction size in isolated hearts subjected to oxidative injury.

Figure 4A shows that submicromolar concentrations of NADH-GA protects cardiomyocytes from damage caused by oxidative stress. The addition of the inhibitor to the perfusate following 40 min of global ischemia also significantly protects mouse hearts from ischemia/reperfusion (I/R) damage (Fig. 4B). Although the precise mechanism leading to the development of tissue injury during I/R is still not fully understood, it is generally accepted that lactic acidosis is one of the factors causing cell injury [32]. During ischemia anaerobic glycolysis becomes the main pathway supplying the cell with energy [33]. The end product of glycolysis, pyruvate, is converted to lactate in the reaction catalyzed by LDH. It is known that during ischemia cardiac myocytes establish a high level of lactic acid and low intracellular pH [34]. The increase in proton concentration initiates a cascade of events leading to apoptosis and cell death in cultured cells including cardiac myocytes [14, 15, 17, 35, 36]. Earlier studies had shown that ANAD-Pyr, a strong inhibitor of LDH, reduced lactate production in crude brain extracts, although the inhibitor was less effective in synaptosomes [9]. The main reason for the minimal effect in synaptosomes is likely associated with the hydrophilic nature of ANAD-Pyr and the low stability of the compound at physiological pH [9]. NADH-GA is also highly polar and very stable under physiological conditions and in contrast to ANAD-Pyr can not be degraded by LDH. This may be the reason that NADH-GA is protective in both cardiomyocytes and in an ex vivo model of I/R injury (see Fig 4). The expected slow rate of NADH-GA passive transport through the cell membrane is a disadvantage, which could reduce the inhibitor efficiency limiting its application as cardioprotective agent. Attachment of hydrophobic residues by covalent modification of the ribose portion of the inhibitor should strongly increase its permeability through the cell membrane and might lead to a increase of the cardioprotective effect of the inhibitor.

The cardioprotective mechanism of NADH-GA may, however, not be directly related to inhibition of lactic acid production since NADH-GA was added during the reperfusion phase. It is known that restoration of blood flow to the ischemic region during I/R leads to a further increase in tissue damage beyond that caused by ischemia alone [37-43]. The damage during the reperfusion phase is due, in large part, to the generation of reactive oxygen species (ROS). It has been demonstrated that ROS concentration is strongly increased in the myocardium during early reperfusion [44-48]. As shown in Fig. 4B, NADH-GA added at the time of reperfusion restores mechanical function and reduces infarct size in a Langendorff perfused mouse heart model. The mitochondrial respiratory chain is an important source of ROS originating during the first minutes of reperfusion. One may speculate that lactic acid accumulated in large quantities during the ischemic phase may serve as a source of reducing equivalents for the respiratory chain, thus contributing to ROS formation. Lactic acid accumulated during ischemia can be converted to pyruvate by LDH (the reverse reaction) and further metabolized by the citric acid cycle yielding NADH. Subsequent oxidation of NADH by mitochondrial respiratory complexes could also lead to ROS formation. Inhibition of LDH by NADH-GA will slow pyruvate formation from lactate and as a result will suppress ROS formation. This would prevent tissue damage caused by free radicals and would be consistent with the protective effect of NADH-GA reported here.

Supplementary Material

01

Initial purification of the inhibitor on a Q-sepharose Fast Flow column:

The inhibitor was prepared as described in the Materials and Methods section. The solution was applied to a 130 ml capacity Q-sepharose Fast Flow column equilibrated with 5 mM potassium phosphate (pH 12). The inhibitor was eluted from the column with a linear 5- 1000 mM potassium phosphate gradient at pH 12 (dashed line) at a flow rate of 2 ml/min. The elution was monitored at 350 nm. The low observed signal-to-noise ratio of the measured absorption is due to the high optical density of the NADH-containing fractions. Fractions eluting from the column were tested for their ability to suppress LDH activity as described in Materials and Methods. The red curve in Fig. 1 represents the relative inhibition efficiency of the fractions. The fractions containing the inhibitor are eluted from the column after authentic NADH at approximately 400-450 mM potassium phosphate. The peak eluting after NADH-GA, and noted in the figure as NADH-OH, is formed from a different NADH derivative which we have previously shown to be a strong inhibitor of NADH:ubiquinone oxidoreductase (Complex I) [1]. The complete purification of NADH-GA was accomplished by taking the fractions represented by the red peak and further purification as described in Materials and Methods.

[1] Kotlyar, A.B., Karliner, J.S., and Cecchini, G. (2005) A novel strong competitive inhibitor of complex I. FEBS Lett. 579, 4861-4866.

5. Acknowledgments

This work was supported by the Department of Veterans Affairs, Office of Research and Development, Biomedical Laboratory Research Division and by NIH grants GM61606 (GC) and HL068738 (JSK). We are grateful to Violetta Kotlyar for her helpful advice in preparation of the inhibitor.

List of abbreviations

LDH
Lactate dehydrogenase
MDH
Malate dehydrogenase
Q1
2,3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone
APAD
3-Acetylpyridine nicotinamide adenine dinucleotide
NAD-Pyr
Nicotinamide adenine dinucleotide-pyruvate
ANAD-Pyr
3-Acetylpyridine nicotinamide adenine dinucleotide-pyruvate
GA
glycolic acid
HRMS
High Resolution Mass Spectroscopy
HRESIMS
High Resolution Electron Spray Ionization Mass Spectra
COSY
Correlated Spectroscopy
TOCSY
Total Correlated Spectroscopy HSQC, Heteronuclear Single Quantum Coherence
HMBC
heteronuclear multiple bond correlation
I/R
ischemia/reperfusion
BDM
butanedione monoxime
HBSS
Hank's Balanced Salt Solution
K-Pi
potassium phosphate

Footnotes

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