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Am J Physiol Heart Circ Physiol 279: H1839-H1848, 2000;
0363-6135/00 $5.00
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Vol. 279, Issue 4, H1839-H1848, October 2000

2,3-Butanedione monoxime unmasks Ca2+-induced NADH formation and inhibits electron transport in rat hearts

Russell C. Scaduto Jr. and Lee W. Grotyohann

Department of Cellular and Molecular Physiology, Penn State College of Medicine, Pennsylvania State University, Hershey, Pennsylvania 17033


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

We used 2,3-butanedione monoxime (BDM) to suppress work by the perfused rat heart and to investigate the effects of calcium on NADH production and tissue energetics. Hearts were perfused with buffer containing BDM and elevated perfusate calcium to maintain the rates of cardiac work and oxygen consumption at levels similar to those of control perfused hearts. BDM plus calcium hearts displayed higher levels of NADH surface fluorescence, indicating calcium activation of mitochondrial dehydrogenases. These hearts, however, displayed 20% lower phosphocreatine levels. BDM suppressed the rates of state 3 respiration of isolated mitochondria. Uncoupled respiration was suppressed to a lesser degree, and the state 4 respiration rates were not affected. Double-inhibitor experiments with liver mitochondria using BDM and carboxyatractyloside (CAT) were used to identify the site of inhibition. BDM at low levels (0-5 mM) suppressed respiration. In the presence of CAT at levels that inhibit respiration by 60%, low levels of BDM were without effect. Because these effects were not additive, BDM does not inhibit adenine nucleotide transport. This was supported by an assay of adenine nucleotide transport in liver mitochondria. BDM did not inhibit ATP hydrolysis by submitochondrial particles but strongly suppressed reversed electron transport from succinate to NAD+. Oxidation of NADH by submitochondrial particles was inhibited by BDM but oxidation of succinate was not. We conclude that BDM inhibits electron transport at site 1.

adenine nucleotide translocase; heart; mitochondria; F0F1-adenosinetriphosphatase; fluorescence; reduced nicotinamide adenine dinucleotide; calcium; bioenergetics; site 1


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE CARDIOPLEGIC AGENT 2,3-butanedione monoxime (BDM) was developed in 1955 as a reactivator of acetylcholinesterase poisoned by organophosphorous compounds (42). This general phosphatase activity was initially thought to be the basis for its negative inotropic action (40). It is now well documented that BDM displays multiple effects in skeletal and cardiac muscle.

Acting as a phosphatase, BDM inhibits the cardiac transient outward potassium current (43) and the L-type calcium channel (8) by dephosphorylation. Although BDM itself may exert direct phosphatase activity, it also activates type 1 and type 2A phosphatases, causing a lowered phosphorylation state of the inhibitory subunit of troponin and phospholamban (46). BDM also causes a release of calcium from cardiac sarcoplasmic reticulum without inhibition of calcium uptake (31, 37). More recently, BDM has also been shown to block rat heart gap junction channels (38). These effects are observed at higher concentrations of BDM (10-30 mM), whereas significant muscle relaxation occurs at lower concentrations.

Excitation-contraction coupling is suppressed at lower concentrations of BDM due to disruption of cross-bridge kinetics (16, 36, 45). This effect is primarily due to BDM increasing the binding constants for MgATP and MgADP (17). At concentrations <10 mM, BDM has little to no effect on the cardiac calcium transient, although contraction is markedly suppressed (10, 29, 30). Because of these results, BDM has also been used to investigate the effects of cellular calcium on cardiac bioenergetics. It allows cellular levels of calcium to be altered without stimulation of contraction-mediated ATP hydrolysis. It has also been used as a cardioplegic agent, where BDM has been shown to enhance the recovery of postischemic cardiac function (27).

There has been evidence to suggest that an elevation in mitochondrial calcium stimulates cardiac respiration. The majority of this evidence, however, has been obtained either indirectly or from studies of isolated mitochondria (5, 23, 25). A direct stimulation of respiration by calcium is difficult to demonstrate because calcium elicits an increased contractility, which can increase respiration by means other than an effect of calcium on mitochondrial dehydrogenase activity.

In this study, we used BDM in the intact perfused rat heart to disassociate the effects of calcium on stimulating ATP utilization from the potential effects on stimulating mitochondrial respiration. By suppressing contractile activity, we could examine more specifically the effect of calcium on respiration. Increasing the perfusion pressure and media calcium concentration of perfused rat hearts elevated mechanical work, oxygen consumption, and time-averaged intracellular calcium. BDM was added to suppress work under these conditions to levels observed in the absence of BDM to establish a preparation with higher intracellular calcium content but with similar rates of work and ATP hydrolysis. Using this approach, we anticipated that the effects of calcium on mitochondrial metabolism could be observed in the absence of the associated increase in mechanical work and ATP turnover.

In the presence of BDM, NADH levels increased in response to the increase in perfusate calcium and pressure, although the rates of oxygen consumption and cardiac work remained at control levels. Levels of phosphocreatine and ATP, however, decreased significantly. We report that BDM can unmask the stimulating effect of mitochondrial calcium on NADH formation but that BDM inhibits respiration at site 1 of electron transport.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Fluorescence measurements. Surface fluorescence of the isolated perfused rat heart was measured using a fluorometer with a surface fluorescence attachment (C&L Instruments). Surface fluorescence was measured using a bifurcated fused silica fiber-optic bundle. An area of ~1 cm in diameter of the left ventricle was illuminated using the common end of the fiber-optic bundle. The emitted fluorescence was collected using the common end of the same fiber-optic bundle and was directed to a photomultiplier tube detector operating in the photon count mode. Further details have been described previously (35).

Surface fluorescence measurements using a 340-to-380-nm excitation fluorescence ratio were used to estimate mitochondrial NADH as previously described (35). Briefly, the left ventricle was illuminated by light alternating between 340 and 380 nm (10-nm bandpass) to excite NADH while the emission was detected at 500 nm (40-nm bandpass). Although these wavelengths also detect NADPH and cytosolic NADH, this measurement is considered to be originating primarily from mitochondrial NADH (26). Moreover, NAD+ represents ~85% of the total NADP+ plus NAD+ pool in heart mitochondria, and only the NAD+ pool responds to changes in calcium and substrate supply (29). Hearts were perfused under control conditions for a 15-min equilibration period before they were changed to an experimental buffer containing BDM. Optical recordings were made 10 min later, although cardiac work and oxygen consumption became stable 3-4 min after switching to the experimental media.

The amount of NAD+ in the reduced form was expressed as a percentage of the range obtained during the calibration process after perfusion of each heart. Maximal reduction was achieved by perfusion with buffer equilibrated with 95% N2-5% CO2 for 10 min. This was followed by perfusion with substrate-free buffer in the presence of 95% O2-5% CO2 for 20 min to obtain maximal oxidation. This calibration procedure was performed in all hearts in which NAD+ reduction was measured. Parallel perfusion experiments were performed for determination of tissue metabolites in which hearts were frozen.

Isolation and incubation of mitochondria. Mitochondria were isolated from the rat heart as described previously (33). Identical buffers and centrifugation protocols were also used to isolate mitochondria from the liver. Incubations were conducted at 28°C in a medium composed of (in mM) 135 KCl, 20 3-(N-morpholino)propanesulfonic acid (MOPS), 5 K2HPO4, and 5 MgCl2 at pH 7.00. Substrates and other agents were added as indicated in the figure legends.

Mitochondrial oxygen consumption was determined at 28°C in a water-jacketed vessel fitted with a Clark electrode and a stirring apparatus. The respiratory-to-control ratios (state 3-to-state 4 respiratory rate ratios) of all preparations were determined. Preparations with control ratios <6 were not used. Isolated mitochondria were kept at 4°C and used within 4 h after isolation.

Adenine nucleotide exchange rates of liver mitochondria were determined as described by Aprille and Asimakis (1). Mitochondria (1 mg/ml) were preincubated for 1 min at 4°C. [14C]ADP (1.6 Ci/mol, 20 nmol/ml) was then added, and the suspension was rapidly vacuum filtered (vacuum manifold, Hoefer Scientific Instruments) through a glass-fiber filter (1 µm, Fisher G4) at either 5, 15, 30, or 45 s after [14C]ADP was added to separate the mitochondria from the media. The filters were washed once with 5 ml of ADP-free media (at 4°C). The filters were assayed for 14C by scintillation counting. Rates of 14C accumulation by mitochondria were linear for up to 1 min. Addition of carboxyatractyloside (CAT, 2 µM) lowered [14C]ADP accumulation by the mitochondria to amounts observed by extrapolation of the data (Fig. 4) to the initial time of [14C]ADP addition.

Mitochondrial membrane potential (Delta Psi ) was determined by measuring the fluorescence wavelength shift of tetramethylrhodamine methyl ester due to a Delta Psi -dependent uptake of this indicator, as described in detail previously (34).

Isolation and incubation of submitochondrial particles. Nonphosphorylating EDTA submitochondrial particles (SMP) were prepared by sonication of the rat heart mitochondria as described by Panov and Scaduto (28). Particles were incubated (0.05 mg/ml) for an assay of ATP hydrolysis using media composed of (in mM) 130 KCl, 40 MOPS, 5 MgCl2, 0.2 EGTA, and 1 ATP at pH 7.40. Reactions were initiated by addition of ATP and were terminated 2 min later with trichloroacetic acid (final concentration, 8%). ATPase activity was measured as the rate of phosphorous accumulation. Inorganic phosphorus was determined colorimetrically using the acid molybdate method as prepared in the phosphorus kit by Sigma Chemical (St. Louis, MO).

Phosphorylating SMP were prepared by sonication of the rat heart mitochondria in a buffer composed of (in mM) 250 sucrose, 15 MgCl2, 10 MOPS, and 1 ATP at pH 7.40; essentially as described by Lee and Ernster (21). These particles were used to assess the rates of reversed electron transport from succinate to NAD+. The rates of NADH formation were measured spectrophotometrically at 340 nm upon incubation of SMP in media composed of (in mM) 225 sucrose, 75 mannitol, 20 MOPS, 5 MgCl2, 5 succinate, 3 ATP, 1.5 KCN, and 1 NAD+ at pH 7.40. The reactions contained 0.25 mg/ml SMP and were initiated by addition of ATP. NAD+ reduction did not occur in the absence of either ATP, succinate, or SMP or in the presence of either oligomycin (1 µg/mg protein) or rotenone (1 µg/mg protein).

NADH dehydrogenase activity of nonphosphorylating SMP was determined by monitoring the reduction of ferricyanide and 2,6-dichloroindophenol (DCIP) spectrophotometrically in a buffer containing (in mM) 145 KCl, 20 MOPS, and 10 K2HPO4 at pH 7.40 (18). Potassium ferricyanide [K3Fe(CN)6] and DCIP were used at concentrations of 0.1 and 0.5 mM, respectively, in the presence of 3.5 µg/mg antimycin A. Ferricyanide reduction was monitored at 420 nm, and DCIP was monitored at 600 nm. Data are expressed as the rate of NADH oxidation.

Isolated perfused rat heart. Isolated rat hearts were perfused at a constant aortic pressure of either 60 or 110 mmHg using the Langendorff procedure as described elsewhere (35). Oxygen consumption of the perfused rat heart was estimated from the coronary flow rate and the change in PO2 of the perfusate as sensed by two flow-through Clark electrode assemblies. The flow rate was measured using a flow transducer in series with the aortic canulla (Transonic Systems). All hearts were electrically paced at 300 beats/min using a Grass stimulator (model S48) synchronized with the fluorescence data acquisition system. Cardiac work was assessed by insertion of a balloon (no. 3 balloon from Ragnoti Glass Technology) into the left ventricle. The balloon was filled with water, connected to a pressure transducer, and adjusted to zero end-diastolic pressure. Voltage measurements from the transducer were collected by the fluorescence data acquisition system and converted to pressure after calibration.

All hearts were perfused for 15 min under control conditions using 60 mmHg aortic pressure in the Langendorff mode. After this period, the perfusate was switched to media containing BDM and/or an altered calcium concentration. All optical and functional measurements were made 10 min after switching to this media. In other experiments, hearts were frozen at this time for measurement of tissue metabolites. We found that the measured parameters became stable within 3-4 min after switching to the new media. Daily experiments conducted either with or without BDM were randomized.

In some experiments, hearts were quickly frozen during the course of perfusion using aluminum clamps previously cooled in liquid N2. Frozen tissue was pulverized under liquid N2, extracted with perchloric acid, neutralized, and assayed for metabolite content. These assays were performed by standard procedures in which the metabolite of interest was linked enzymatically to the appearance or disappearance of NADH. The extraction and assays were performed as previously described (33, 39). The levels of free ADP were estimated from the following: the creatine kinase equilibrium (Keq = [ATP][Cr]/[ADP][H+][PCr], where Keq is the equilibrium constant, Cr is creatine kinase, and PCr is phosphocreatine) of 1.66 × 10-9 (20), the measured ATP and phosphocreatine content, a cytosolic pH of 7.05 (9), a cytosolic volume of 3.23 ml/g dry wt (14), and a total phosphocreatine plus creatinine content of 68.5 µmol/g dry wt (41). Any errors in the constants used for this calculation would only affect the estimated concentration of the free ADP concentration but not the conclusions based on changes in free ADP levels between experimental and control groups.

Reagents and chemicals. All chemicals were purchased from Sigma Chemical. For some experiments, lots of BDM were purified by sublimation. Sublimation was performed at 70°C over a period of 12 h using tap water to cool the condensing finger.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of BDM on metabolism of perfused rat heart. Hearts were perfused with concentrations of BDM ranging from 0-10 mM to determine the effectiveness of BDM in suppressing calcium and pressure-induced increases in myocardial oxygen consumption and cardiac work. These data (not shown) indicated that the rates of myocardial oxygen consumption were doubled by increasing the perfusate concentration of calcium from 1.25 to 2.5 mM and increasing perfusion pressure from 60 to 110 mmHg. Hearts perfused with media containing 2.5 mM calcium and between 7.5 and 8.5 mM BDM at 110 mmHg, however, displayed rates of oxygen consumption and work that were similar to those of control hearts perfused with media containing 1.25 mM calcium at 60 mmHg in the absence of BDM. Figure 1 illustrates the effects of 8.5 mM calcium on myocardial work, oxygen consumption, and NADH levels. In the presence of BDM, the rates of oxygen consumption and work at elevated concentrations of calcium and pressure were not different from hearts perfused with control levels of calcium at 60 mmHg. The percentage of reduction of mitochondrial NAD+, however, was increased from 31% to 45%. This increase in the level of NADH can be ascribed to the effects of increased calcium on activation of calcium-sensitive mitochondrial dehydrogenases (12, 22). In the absence of BDM, calcium-mediated elevation of cardiac work causes a decrease in steady-state NADH levels (35). Without BDM, the elevated level of calcium stimulates NADH utilization and the net effect results in a lowered level of NADH. Hence, the elevated level of NADH in these experiments, in which NADH utilization is limited by the presence of BDM (Fig. 1), can be viewed as unmasking the effects of calcium on mitochondrial NADH generation.


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  		Fig. 1.   Effect of 2,3-butanedione monoxime (BDM) on cardiac work, O2 consumption, and NADH levels. Hearts were perfused with buffer containing 1.25 mM calcium at 60 mmHg (low pressure) or with buffer containing 2.5 mM calcium plus 8.5 mM BDM at 110 mmHg (high pressure). Plotted are the peak ventricular pressure (Peak VP, in mmHg; hatched bars), maximum change in pressure over time (max dP/dt, in mmHg/s; crosshatched bars), O2 consumption (O2 Cons., in µmol/min; open bars), and percentage of NAD+ reduction (% Red.; shaded bars). In the presence of BDM, increases in perfusate calcium and perfusion pressure did not increase cardiac work, although levels of NADH were higher in the presence of BDM. *P < 0.05; n = 4 hearts.

In parallel experiments, hearts were perfused in a fashion similar to the experiments illustrated in Fig. 1, except that the tissue was frozen for metabolite assays. The total content of ATP, ADP, AMP, and phosphocreatine, as well as the calculated free ADP concentration in these hearts, is shown in Fig. 2. Hearts perfused with higher pressure in the presence of BDM and elevated levels of calcium displayed significantly depressed levels of ATP and phosphocreatine. As a result, the calculated tissue concentration of free ADP increased ~50%. Similar results were obtained in experiments using 7.5 mM BDM (data not shown). Because these effects on ATP and phosphocreatine occurred despite elevated levels of NADH and equivalent rates of oxygen consumption (Fig. 1), the data indicated that BDM might suppress a process of oxidative phosphorylation distal to the generation of NADH.


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  		Fig. 2.   Effect of BDM on cardiac energetics. Hearts were perfused as described in METHODS and in Fig. 1. The tissue levels of ATP (hatched bars), ADP (crosshatched bars), AMP (open bars), phosphocreatine (PCr, shaded bars), and the calculated free ADP concentration (ADPfree, solid bars) are shown. ADPfree was calculated as described in METHODS. In the presence of BDM, increases in perfusate calcium and perfusion pressure decreased levels of ATP and PCr while elevating levels of ADPfree. *P < 0.05; n = 4 hearts.

Effects of BDM on metabolism of isolated mitochondria and SMP. To further investigate the effects of BDM on cardiac bioenergetics, experiments were performed with isolated heart mitochondria. With glutamate and malate as substrates, the effect of BDM on mitochondrial respiration is shown in Fig. 3. BDM strongly suppressed ADP-stimulated respiration, but it had no effect on state 2 respiration in the absence of ADP (Fig. 3A). It also had no effect on state 4 rates of respiration (data not shown). In other experiments, respiration was progressively stimulated with the uncoupler 2,4-dinitrophenol (DNP). BDM was considerably less effective in suppressing DNP-stimulated respiration (Fig. 3B). In experiments with pyruvate plus malate as substrates, BDM similarly suppressed state 3 respiration. With this substrate combination, the state 3 rates were inhibited 47% and uncoupled respiration rates were inhibited 23% by 10 mM BDM (data not shown).


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  		Fig. 3.   Effects of BDM on respiration of heart mitochondria. Rat heart mitochondria (0.75 mg/ml) were incubated with 10 mM glutamate and 5 mM malate as substrates as described in METHODS. BDM was included at the concentration of 0 (), 2.5 (black-triangle), 5 (black-lozenge ), and 10 mM (). The steady-state rates of O2 consumption when respiration was stimulated with either ADP (A) or 2,4-dinitrophenyl (DNP; B) are shown. In A, the media also contained 20 mM glucose and 7 IU/ml hexokinase.

The effects of BDM on respiration were also investigated by monitoring steady-state NADH fluorescence under conditions of ADP and uncoupler-stimulated respiration. Stimulation of cardiac respiration is known to decrease steady-state levels of NAD(P)H (35), but BDM did not affect the response of NAD(P)H to ADP (data not shown). The levels of NAD(P)H at low levels of ADP, however, were lower than control levels at all concentrations of BDM. The cause of this effect was unresolved but could have been due to an influence of BDM as an inner filter of the measured fluorescence because BDM absorbs light in the region of NADH absorbance (data not shown). Similar results were observed using DNP to stimulate respiration (data not shown).

These data suggest that BDM inhibits oxidative phosphorylation at a step distal to the generation of NADH. This inhibition pattern suggests that BDM could suppress generation of mitochondrial Delta Psi by inhibition of electron transport or utilization of mitochondrial Delta Psi by ATP synthesis. ATP synthesis could be suppressed by inhibition of either the ATP synthase or adenine nucleotide transport. Because it has been reported that BDM inhibits adenine nucleotide exchange in mitochondria from skeletal muscle (24), we used liver mitochondria to directly measure adenine nucleotide exchange. Preliminary experiments illustrated that BDM also suppressed the respiration of mitochondria from rat livers (data not shown) in a fashion similar to that observed with heart mitochondria. Mitochondria from rat hearts were not used because the exchange rates in heart mitochondria are too rapid to monitor with this method (19).

Mitochondria from the liver were incubated at 4°C in the presence of [14C]ADP and BDM to determine the rates of adenine nucleotide exchange. Figure 4 illustrates that ADP exchange by liver mitochondria was not affected by BDM at concentrations up to 10 mM.


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  		Fig. 4.   Adenine nucleotide exchange in liver mitochondria. Rat liver mitochondria (1 mg/ml) were incubated as described in METHODS. [14C]ADP (20 µM) was added, and the mitochondria were separated from the media by vacuum filtration at the indicated times. BDM was included at the concentration of 0 (), 5 (black-triangle), and 10 mM (). Shown is the mitochondrially associated 14C activity.

To determine whether BDM inhibits ATP synthase, a double-inhibitor experiment was performed. It is well recognized that CAT is a potent and specific inhibitor of the adenine nucleotide transporter in the inner mitochondrial membrane. Furthermore, the rates of respiration can be titrated with CAT and, in the presence of CAT, ADP-stimulated respiration is limited primarily by the rate of this transporter (6).

In control mitochondria, increasing concentrations of BDM between 0 and 10 mM progressively suppressed the rates of state 3 respiration (Fig. 5). A titration with CAT over a range of 0-0.4 µM showed a similar effect (data not shown). In the presence of 0.2 µM CAT, state 3 respiration of mitochondria was inhibited by 60%. In the presence of 0.2 µM CAT, however, BDM in the concentration range between 0 and 4 mM was essentially without effect (Fig. 5). Because the effects of BDM and CAT were not additive, the primary site of BDM inhibition is not in the adenine nucleotide transporter. If, however, BDM suppressed adenine nucleotide exchange, low levels of BDM would be expected to affect respiration in the presence of CAT because this exchange carrier limits respiration under these conditions.


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  		Fig. 5.   Effect of carboxyatractyloside on BDM-mediated inhibition of ADP-stimulated respiration. Rat heart mitochondria were incubated with 1 mM pyruvate and 5 mM malate as substrates. State 3 respiration was attained by addition of 1 mM ADP. Addition of BDM progressively inhibited respiration (). BDM did not suppress respiration to the same extent in the presence of 0.2 µM carboxyatractyloside (black-triangle), especially at low levels of BDM.

We also tested the sensitivity of state 3 respiration of intact mitochondria to the concentration of phosphate because phosphate is required for ADP-stimulated respiration. In the presence of BDM, the maximal rates of ADP-stimulated respiration were strongly suppressed. Thus the apparent the maximal velocity rate (Vmax) for phosphate was also lowered by BDM. The apparent Michaelis-Menten constant for phosphate, taken as the concentration that gave one-half of the Vmax respiration rate, was not affected by BDM at concentrations up to 10 mM (data not shown).

To assess the effects of BDM on ATP synthase, the rates of ATP hydrolysis were measured in SMP. ATPase activity was measured as the rate of phosphate accumulation. BDM in the range of 0-10 mM did not have any effect on ATP hydrolysis by either nonphosphorylating or phosphorylating SMP (data not shown). These data are in agreement with those obtained previously with mitochondria from skeletal muscle (24).

To determine the effects of BDM on electron transport, the rates of electron transport were measured in SMP. Forward electron transport from NADH to oxygen was inhibited by BDM, whereas the rates from succinate to oxygen were not (Fig. 6). These data suggest that BDM inhibits electron transport at site 1. To confirm this observation, the rates of reversed electron transport from succinate to NAD+ were determined. Figure 7 illustrates that BDM inhibited NAD+ reduction by SMP incubated with succinate and ATP.


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  		Fig. 6.   Effect of BDM on O2 consumption by submitochondrial particles. Nonphosphorylating submitochondrial particles were incubated as described in METHODS, and O2 consumption was determined with a Clark electrode. Shown are rates obtained with either 5 mM succinate () or 1.5 mM NADH (black-triangle) as substrate. Data represent the average of duplicate reactions.



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  		Fig. 7.   Effect of BDM on reversed electron transport by submitochondrial particles. Phosphorylating submitochondrial particles were incubated as described in METHODS. Rates of NAD+ reduction were measured as NADH formation at 340 nm. The mean rates ± SE from 3-5 incubations are shown.

In other experiments, artificial electron acceptors were used to determine the effects of BDM on NADH dehydrogenase. In the presence of either ferricyanide or DCIP, this enzyme will pass electrons to the acceptor rather than into site 1 (18). The results shown in Table 1 illustrate that the reduction of potassium ferricyanide or DCIP by SMP incubated with NADH was not affected by BDM, whereas the oxidation of NADH with oxygen as electron acceptor was strongly inhibited.

                              
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  		Table 1.   Effect of BDM on NADH dehydrogenase activity of submitochondrial particles

If BDM inhibits electron transport, then BDM should lower the membrane potential of intact mitochondria under conditions of stimulated respiration. Table 2 shows the mitochondrial Delta Psi upon incubation of mitochondria with pyruvate plus malate or with succinate as substrate(s). BDM lowered mitochondrial Delta Psi with pyruvate plus malate as substrates but not with succinate as a substrate. The control mitochondrial Delta Psi obtained with pyruvate plus malate as substrates was greater than the mitochondrial Delta Psi obtained with succinate. This was expected because the rates of respiration in heart mitochondria are also lower with succinate. In other control experiments, either antimycin A or oligomycin was used at concentrations sufficient to suppress state 3 respiration by about 50%. Consistent with the site of action of these agents, antimycin A lowered mitochondrial Delta Psi under state 3 respiration, whereas oligomycin elevated mitochondrial Delta Psi (data not shown). The lowering of mitochondrial Delta Psi by BDM also indicates that BDM, like antimycin A, suppresses respiration at a site proximal to the generation of mitochondrial Delta Psi . This provides additional evidence that the primary site of BDM inhibition is in electron transport and not in the adenine nucleotide translocase or mitochondrial ATPase.

                              
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  		Table 2.   Effect of BDM on mitochondrial membrane potential

Purity of BDM. We noted that the commercial preparations of BDM appeared to contain varying amounts of a yellow contaminant. To determine whether this impurity was the cause of the inhibitory effects of BDM, BDM was purified by sublimation as described in METHODS. BDM purified in this manner had a pure white appearance but exhibited identical inhibitory properties to the crude commercial material. The purified BDM was used in all experiments except for those indicated in Fig. 3. The purity of the original material was estimated to be ~95% (g/g) because ~5% of the original material remained after prolonged sublimation.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A primary effect of an elevation of cytosolic calcium in the heart is the stimulation of muscle contraction and myosin ATPase activity. This effect of calcium increases, in parallel, both ATP hydrolysis in the cytosol and ADP phosphorylation in the mitochondria. Increases in contractile activity in the heart, however, are not associated with detectable changes in the levels of phosphocreatine or adenine nucleotides (3). These observations led to the suggestion that factors other than the availability of free ADP control respiration in this tissue. To explain these observations, it has been proposed that elevation in cytosolic calcium leads to elevations in mitochondrial calcium, which in turn activate mitochondrial dehydrogenases and pyruvate dehydrogenase phosphatase (12). A direct effect of calcium in increasing mitochondrial NADH, consistent with activation of dehydrogenase activity, has been observed in isolated mitochondria (11, 29). It remains unclear, however, whether the calcium-mediated increase in NADH participates in the control of cardiac respiration. In isolated mitochondria, the maximal rates of respiration are higher in the presence of calcium (25), and calcium causes a more sustained membrane potential as respiration is increased from state 4 to state 3 (29). More recently, calcium has been shown to increase the control coefficient exerted by the dehydrogenases in studies of the rate control of coupled oxidative phosphorylation (23).

In intact tissue, the importance of elevations in mitochondrial NADH as a control factor is less clear. Increases in cardiac work, in fact, have been shown to cause a decrease in NADH in studies of myocytes, rat trabeculae, and the perfused heart (2, 15, 32). This suggests that these processes may not be significant, but it is also unclear what proportion of this level of NADH is maintained by an enhanced dehydrogenase activity under conditions of increased flux. That is, the levels of NADH under conditions of stimulated work may decrease to even lower values if not for the effect of calcium in stimulating these dehydrogenases.

One difficulty in studying the effects of calcium on mitochondrial metabolism in intact preparations is the ability to distinguish between the primary and secondary effects of calcium. To dissociate these effects, we used BDM in this study to suppress the effects of calcium on stimulation of contraction and myosin ATPase activity. These experiments in the perfused heart were designed to establish a preparation in which tissue levels of free calcium could be elevated without eliciting the effect of calcium in stimulation of myocardial oxygen consumption. BDM has been used previously for this purpose in a study of the effect of calcium on mitochondrial Delta Psi (7). At low concentrations (<10 mM), BDM does not affect the cytosolic levels of calcium in the heart (10, 29, 30). In the present work, the concentration of BDM in the perfusate was adjusted to maintain control levels of mechanical work and oxygen consumption when levels of calcium were increased. These hearts displayed elevated levels of NADH, which is consistent with a Ca2+-mediated activation of mitochondrial dehydrogenases. It should be noted that in the absence of BDM, an elevated level of perfusate calcium normally causes NADH levels to decrease because NADH consumption is also stimulated (35). Thus the use of BDM in this manner unmasks the effect of calcium on increasing mitochondrial NADH formation. Jiang and Julian (15) observed a similar result. These authors observed that NADH fluorescence of rat heart trabeculae was increased by pacing in the presence of BDM, whereas pacing decreased NADH in the absence of BDM.

Despite the fact that NADH levels increased when calcium availability was increased in the presence of BDM, the levels of ATP and phosphocreatine decreased significantly (Fig. 2). This effect of BDM in the heart had not been described previously. The opposite effect would have been predicted based solely on the ability of BDM to increase NADH under these conditions. Of additional interest was the fact that the ability of BDM to decrease phosphocreatine and ATP levels was not observed with higher concentrations of this inhibitor (data not shown). Presumably, with higher BDM concentrations, mitochondria are respiring at near state 4 rates, and the residual activity of ATP synthase is sufficient to maintain the levels of phosphocreatine and ATP under these low work conditions. This is in keeping with our observations that BDM did not affect rates of state 4 respiration of isolated mitochondria.

A prior study (24), discussed in more detail below, suggested that BDM inhibits mitochondrial adenine nucleotide translocase. We examined the role of this translocase in heart mitochondria using double-inhibitor experiments with CAT. CAT specifically inhibits mitochondrial adenine nucleotide translocase. At concentrations of CAT that suppress respiration by 60%, low concentrations of BDM did not suppress respiration further. Because it is well established that in the presence of CAT, the limited activity of adenine nucleotide translocase renders the translocase the major "rate-controlling" step of respiration (6), these data indicate that BDM does not inhibit the translocase. If BDM did inhibit translocase activity, low BDM concentrations would be expected to exert a marked effect under these conditions in which the translocase activity is limiting respiration. In fact, the opposite effect was observed. BDM was less effective when CAT suppressed respiration. This observation suggests that BDM inhibits a site other than the translocase. Direct measurement of the translocase activity in liver mitochondria (Fig. 4) supported this conclusion.

Other data obtained with SMP illustrate that BDM inhibits site 1 of electron transport. NADH oxidation using oxygen as an electron acceptor was inhibited by BDM, whereas succinate oxidation was not. Moreover, reversed electron transport (ATP supported NAD+ reduction by succinate) was also inhibited. Thus respiration of the perfused heart can be suppressed by BDM by at least two mechanisms. The first mechanism is the documented effect of suppressing myosin ATPase activity, and the second mechanism is due to the inhibition of electron transport. At the lower concentrations of BDM used in this study, ATP and phosphocreatine levels decreased when respiration rates were maintained by an increase in perfusate calcium. Under these conditions, it is possible that BDM suppressed respiration by inhibiting both sites. The relative influence of these two mechanisms under the conditions used in this study cannot be evaluated without further investigation. It must be emphasized that in these experiments, however, BDM lowered ATP and phosphocreatine levels and increased NADH levels when the rates of oxygen consumption were maintained by an increase in perfusate calcium. This indicates that flux through electron transport was similar under all conditions. It is likely that electron transport flux and oxygen consumption were maintained by the elevated level of calcium via activation of mitochondrial dehydrogenases and/or the elevated concentration of ADP.

Because the rates of oxygen consumption were maintained by the elevation of perfusate calcium in perfusions containing BDM, the increase in NADH under these conditions in the intact heart must have occurred via activation of mitochondrial dehydrogenases. The increase in NADH could not have resulted from the inhibition of electron transport, because flux through electron transport was maintained constant by adjusting the calcium levels. In addition, this observation could not have been from the result of a stimulation of glycolysis, because measurement of NADH by surface fluorescence is considered an index of mitochondrial NADH (26). Steady-state NADH levels are determined solely by the balance between its consumption by electron transport and generation by mitochondrial dehydrogenases. This is the essence of metabolic regulation and the means by which the concentration of metabolic intermediates are established, as reviewed by Brand (4). Inhibition of the electron transport by BDM cannot explain our observation of an increase in NADH in perfused hearts because the respiration rates were maintained at control levels. The consistency in the rates of oxygen consumption indicate that NADH consumption was similar in control and BDM-treated hearts. These observations are distinct from those that would be caused by either oligomycin or hypoxia. With either oligomycin or hypoxia, NADH levels increase because both electron transport and NADH consumption are suppressed.

Other potential modes of BDM action can be ruled out. In the absence of elevated perfusate calcium, BDM strongly inhibits cardiac work and oxygen consumption by the perfused heart. In addition, BDM did not increase the state 4 respiration of isolated mitochondria, which indicates that BDM does not uncouple oxidative phosphorylation. For the reasons discussed above, it is also unlikely that the increase in NADH in hearts perfused with BDM (plus additional calcium) is due to the inhibitory effect of BDM on electron transport. The increase in NADH that would occur with anoxia or by treatment with oligomycin would be caused by a decrease in NADH consumption, which was prevented in the present study by elevation of perfusate calcium.

In the isolated liver (data not shown) and cardiac mitochondria (Fig. 3), ADP-stimulated respiration was affected to a greater degree than was uncoupler-stimulated respiration. This was more evident with glutamate plus malate as substrates. Uncoupled respiration with pyruvate plus malate as substrates, however, was significantly inhibited by BDM. This observation can be explained by the fact that mitochondrial Delta Psi is required for glutamate transport via the electrogenic glutamate-aspartate exchange carrier. Thus overall rates of respiration are lower with this substrate combination under uncoupled conditions. This can account for the lower effects of BDM observed under uncoupled conditions in incubations with glutamate.

It should be pointed out that a prior communication (24) suggested that BDM inhibits adenine nucleotide translocase. These authors concluded that this action may be an addition mechanism contributing to the lowered twitch tension in skeletal muscle treated with BDM. It was recognized, however, that the effect of BDM on mitochondrial metabolism may not lower tissue ATP levels, because the utilization of ATP by the contractile apparatus is strongly suppressed by BDM. This is in accordance with our observations in the heart. We observed that higher levels of BDM did not affect tissue phosphocreatine and ATP levels, presumably because the demand for ATP was considerably lower under these conditions. Tissue phosphocreatine and ATP levels were decreased only when the rates of respiration were maintained by elevation of the perfusate calcium concentration (Fig. 2).

The site of BDM inhibition proposed by the prior authors (24) was based on the observation that ADP-mediated respiration, but not uncoupled respiration, was inhibited by BDM and that the ATPase activity of SMP was unaffected by BDM. These authors did not measure translocase activity directly. Using liver mitochondria, we found that translocase activity was not affected by BDM (Fig. 4). These authors dismissed a possible action of BDM on electron transport because uncoupled rates of respiration were unaffected by BDM. These prior studies, however, were conducted with succinate as a substrate and, thus, an inhibition by BDM at site 1 of electron transport would not have been as apparent with this substrate. Of interest, however, is the observation by these authors that there is a minor inhibition of ADP-stimulated respiration with succinate as a substrate (~15% inhibition at 10 mM BDM). In similar experiments with heart mitochondria, we did not observe inhibition of respiration with succinate as a substrate (data not shown). Although other less significant sites of BDM inhibition of mitochondrial respiration may exist, our data indicate that the major inhibitory site is in site 1 of electron transport. We found that BDM caused a decrease in mitochondrial Delta Psi under conditions of ADP-stimulated respiration (Table 2). This indicates that reactions generating mitochondrial Delta Psi are suppressed to a greater extent by BDM than those consuming mitochondrial Delta Psi . Thus the primary site of BDM inhibition cannot be the translocase.

BDM has been used to estimate the nonmechanical component of oxygen consumption in the heart (13, 44). Higher levels of BDM (>10 mM) strongly suppress cardiac respiration. Our data suggest that measurement of nonmechanical oxygen consumption made in this manner may be underestimated as a result of BDM inhibition of mitochondrial metabolism.

In summary, our studies with isolated mitochondria show that BDM primarily inhibits oxidative phosphorylation at the level of site 1 of electron transport. Half-maximal inhibition of state 3 respiration of isolated heart mitochondria occurred at a concentration of 5 mM. State 4 respiration rates were unaffected. BDM, therefore, should be classified as a mitochondrial site 1 inhibitor and results obtained with this compound should be evaluated in this light. This inhibitory effect of BDM is unfortunate because it would be a more powerful tool to investigate the effects of calcium in cardiac energetics if it did not inhibit mitochondrial metabolism. Nonetheless, our observations using this inhibitor provide the first evidence obtained in the intact heart that calcium directly stimulates mitochondrial dehydrogenases to elevate matrix NADH levels.


    ACKNOWLEDGEMENTS

We thank Dr. Momcilo Miljkovic for advice and assistance in the purification of 2,3-butanedione monoxime.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-54827.

Address for reprint requests and other correspondence: R. C. Scaduto, Jr., Dept. of Cellular and Molecular Physiology, Milton Hershey Medical Center, Hershey, PA 17033 (E-mail: rscaduto{at}psu.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 26 April 1999; accepted in final form 1 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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