Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061
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ABSTRACT |
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The role of Ca2+ as a cytosolic signaling molecule between porcine cardiac sarcoplasmic reticulum (SR) ATPase and mitochondrial ATP production was evaluated in vitro. The Ca2+ sensitivity of these processes was determined individually and in a reconstituted system with SR and mitochondria in a 0.5:1 protein-to-cytochrome aa3 ratio. The half-maximal concentration (K1/2) of SR ATPase was 335 nM Ca2+. The ATP synthesis dependence was similar with a K1/2 of 243 nM for dehydrogenases and 114 nM for overall ATP production. In the reconstituted system, Ca2+ increased thapsigargin-sensitive ATP production (maximum ~5-fold) with minimal changes in mitochondrial reduced nicotinamide adenine dinucleotide (NADH). NADH concentration remained stable despite graded increases in NADH turnover induced over a wide range of Ca2+ concentrations (0 to ~500 nM). These data are consistent with a balanced activation of SR ATPase and mitochondrial ATP synthesis by Ca2+ that contributes to a homeostasis of energy metabolism metabolites. It is suggested that this balanced activation by cytosolic Ca2+ is partially responsible for the minimal alteration in energy metabolism intermediates that occurs with changes in cardiac workload in vivo.
adenosine 5'-triphosphate; energy metabolism; calciumadenosinetriphosphatase; reduced nicotinamide adenine dinucleotide; porcine heart
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INTRODUCTION |
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THE HEART IS CAPABLE of maintaining the concentration of metabolic intermediates involved in energy conversion over a wide range of metabolic rates. Numerous studies in animal models (3, 11, 19, 22, 25, 26, 29) and in humans (23, 30, 40) demonstrated that the concentrations of ATP as well as creatine phosphate (CrP), inorganic phosphate (Pi) and calculated ADP can remain constant even when the turnover of these molecules changes three- to fourfold. Similarly, studies in model systems demonstrated that the mitochondrial concentration of reduced nicotinamide adenine dinucleotide (NADH) is also maintained during physiological modifications of workload (15, 38, 39). These results suggest that many of the metabolites involved in energy metabolism are held constant in a "metabolic homeostasis" despite large changes in workload. The homeostasis of energy metabolism intermediates may be necessary because energy metabolism proceeds in concert with many other cellular processes. Several energy metabolism metabolites are involved in synthetic and catabolic pathways as well as in other forms of cell signaling and in several aspects of transcription. Thus maintaining a near constant level of these metabolites during normal variations in energy demands, or flux, might be important to the overall homeostasis of cardiac cells.
The stability of these metabolic intermediates in heart cells makes it difficult to justify metabolic control models that rely solely on the simple feedback of metabolic intermediates to control energy flux. On the basis of this realization, other metabolic control models for the heart have been proposed, which include intracellular compartmentation (1, 21, 37), highly cooperative kinetics (20), or parallel activation of metabolism and cellular work with an independent cell signaling system (34, 36).
Ca2+ is a likely candidate as an independent cytosolic
signaling molecule between aerobic metabolism and myocardial work.
Ca2+ regulates two of the major ATPase reactions in the
myocyte, i.e., myosin ATPase and the Ca2+-ATPase of the
sarcoplasmic reticulum (SR). On the energy metabolism side,
Ca2+ can regulate several calcium-sensitive dehydrogenases
(CaDH) (8, 13). Ca2+ can also directly
regulate the ATP hydrolysis activity of the F0/F1-ATPase in vitro (14) and
potentially in vivo (31). Recently, it was shown that
Ca2+ can also activate the
F0/F1-ATPase ATP synthetic activity in cardiac
mitochondria (36). The mechanism of Ca2+
activation of the F0/F1-ATPase activity,
whether direct or through other matrix-membrane interactions, is
unknown. These combined metabolic effects of Ca2+ result in
a balanced activation of both the CaDH that generates the mitochondrial
proton motive force () and the
F0/F1-ATPase that utilizes
to produce
ATP. This combined effect of Ca2+ has been proposed to have
a minimal impact on mitochondrial metabolic intermediates when
Ca2+ is introduced with an increase in energy demand
(34). These effects of Ca2+ on cytosolic
ATPase activity and on ATP synthetic pathways make Ca2+ a
plausible candidate as the cytosolic signaling molecule between these
two processes that would not require large changes in metabolic intermediates in the cytosol or mitochondria.
The purpose of this study was to evaluate the role of Ca2+ concentration ([Ca2+]) in the interaction between the SR Ca2+-ATPase and mitochondrial ATP synthetic activities. The SR Ca2+-ATPase is believed to make up a significant fraction of the total ATPase activity in heart cells, varying from 20% to >50% depending on the myocardial workload (33). In addition, it has been shown that the SR and mitochondria are in close physical proximity in heart cells (for example, see Ref. 41), which is consistent with a tight metabolic coupling between these two organelles. In this study we have attempted to reconstitute SR-mitochondria interactions in vitro by mixing purified porcine SR with mitochondria and evaluating the effect of [Ca2+] on ATP production rates and metabolic intermediates.
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MATERIALS AND METHODS |
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Porcine heart mitochondria. Porcine heart mitochondria were prepared from in situ perfused porcine hearts according to previously published procedures (10, 36).
Porcine heart SR. Porcine cardiac SR was prepared following Reddy et al. (28) with minor modifications. The heart muscle from an anesthetized pig was collected in an identical manner as the mitochondria preparation (10) with an ice-cold in situ perfusion with a (in mM) 280 sucrose, 10 HEPES, and 0.2 K2EDTA solution. Two 40-g sections of left ventricular muscle were cut into 0.5-cm3 pieces and homogenized in 200 ml of ice-cold buffer A [in mM: 30 Tris-maleate, 300 sucrose, 0.5 dithiothreitol (DTT), and 3 NaN3, pH 7.0] with a 250-ml VirTis container and open blades (VirTis model 750) with three 15-s cycles at full speed separated by intervals of 15 s. The SR was isolated from this homogenate with differential centrifugation as previously described (27). The final pellet containing SR vesicles was collected carefully in 1 ml of buffer B (in mM: 10 Tris · HCl, 300 sucrose, and 100 KCl, pH 7.0). The SR fraction was resuspended with a pestle tissue grinder and stored in 0.5-ml aliquots after quick freezing in liquid nitrogen. Protein content was determined with the Bio-Rad assay with BSA as the standard. The typical yield was 30-40 mg protein of SR.
Ca2+-ATPase activity of SR vesicles was determined by measuring the rate of phosphate liberation with the malachite green reagent method (16) as modified by Lanzetta et al. (24) in a medium appropriate for isolated mitochondria. The reaction was started by adding ATP (3 mM final concentration) to 200 µl of buffer C (in mM: 125 KCl, 20 HEPES, 15 NaCl, 5 MgCl2, 1 K2EDTA, 1 EGTA, 2 NaPO4, and 0.1 K+-malate, pH 7.0) containing 5-10 µg of SR, and was stopped after 1-5 min by adding malachite green. The maximum rate of Ca2+-sensitive ATPase was 1,976 ± 180 nmol Pi · minCytochrome aa3 content. Mitochondria concentration was rapidly determined from the cytochrome aa3 (Cyta) content with simple spectrophotometric methods (4).
Mitochondrial protocols. Simultaneous estimates of mitochondrial ATP production, based on oxygen consumption, and [NADH], based on scattering corrected by 450-nm fluorescence, were conducted in buffer C at 37°C with the addition of 3.4 mM Na2ATP as previously described (18). In this study, the rate of respiration was assumed to be proportional to the ATP synthetic rate with an ATP-to-O2 ratio of 5.6. Mitochondria were depleted of Ca2+ by incubating in buffer C for 6 min without a carbon substrate source. Subsequent addition of carbon substrates, ATPases, or SR preparations was assumed to be in the presence of a nominal zero [Ca2+] condition. Free [Ca2+] was calculated as described previously (36).
SR additions, up to 1 g/l, had a small effect on net ATP production in mitochondria (1.0 µmol Cyta/l) incubated in buffer C with 3.4 mM ATP in the absence of Ca2+ or carbon substrates [i.e., no glutamate and malate (G/M)]. A 0.5 g/l addition of SR caused the mitochondrial respiratory rate to increase from 9.1 ± 1.0 to 16.1 ± 0.9 nmol O2 · minData and statistical analysis. Data processing for optical spectra were performed with custom programs written in IDL (v5.2, Research Systems). Details on these spectral fitting procedures were published previously (10, 36).
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RESULTS |
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Effects of steady-state [Ca2+] on SR ATPase, mitochondrial CaDH, and ATP production. SR ATPase activity was determined on isolated SR as described in MATERIALS AND METHODS. CaDH activity was estimated by monitoring the effects of Ca2+ on [NADH] in mitochondria incubated in buffer C supplemented with 5 mM G/M in the absence of ADP or exogenous ATPase (so-called state 4; Ref. 7). Under these conditions of minimal flux, the steady-state changes in [NADH] are assumed to be due to alterations in CaDH activity. The effect of [Ca2+] on overall mitochondrial ATP production capacity was determined on the maximum rate of ATP production (assuming 5.8 ATP/O2) driven by a Ca2+-insensitive ATPase, apyrase (EC 3.6.1.5; Sigma A7646) at 3.0 U/ml.
The results of these studies are summarized in Fig. 1. The data are normalized to the maximum rates or [NADH] for ease of comparison. The SR ATPase maximum activity was 745 ± 19 (n = 7) nmol ATP · min
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(1) |
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(2) |
Metabolic effects of Ca2+ activation of SR-mitochondria reconstituted system. To establish the metabolic effects of adding ADP and Pi alone, a Ca2+-insensitive ATPase, apyrase, was added in the presence of ATP and optimal [Ca2+]. This was done to contrast the effects of ADP and Pi delivery alone with the added effects of Ca2+ activation of metabolism. For the apyrase experiments, mitochondria were incubated in buffer C with (in mM) 3.4 ATP, 492 free Ca2+, and 5 G/M. The apyrase concentration was varied from 0 to 1,500 U/l to generate different levels of mitochondrial ATP production.
For the SR-mitochondria reconstituted system the ratio of SR protein to Cyta was 0.5 (mg protein/nmol Cyta; see MATERIALS AND METHODS) as determined from earlier experiments (see Effect of SR-to-mitochondria ratio). The preincubation was in buffer C with 3.4 mM ATP but without Ca2+. The free [Ca2+] was then increased (0 to 492 nM) by injection of Ca2+ into the chamber. The effects of adding apyrase or Ca2+ on [NADH] and respiration in the two systems are illustrated in Fig. 2 for a paired experiment on the same population of mitochondria. The addition of 5 mM G/M caused a sustained increase in [NADH] in both systems. The presence of Ca2+ resulted in a higher [NADH] in the apyrase system compared with the SR-mitochondria reconstituted system because of the activation of CaDH by Ca2+ in the preincubation medium (36). The addition of apyrase increased respiration as the mitochondrial ATP production was matched to the ATPase activity. This increase in ATP synthetic activity was associated with a net decrease in [NADH], which is consistent with the oxidation of NADH initially exceeding the production rate until a new steady state is attained. In contrast, the addition of Ca2+ to the SR-mitochondria preparation caused a small increase in [NADH] with an increase in ATP turnover generated by the activation of the SR ATPase. The difference between the Ca2+-activated ATPase activity in the SR-mitochondria preparation and the simple increase in ATPase activity on mitochondrial [NADH] is proposed to be due to the balanced activation of metabolism and ATPase activity with the Ca2+ addition.
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Effect of thapsigargin.
To ensure that the effects of Ca2+ on ATP production were
due to SR ATPase activity, thapsigargin was used as an inhibitor
of SR ATPase activity. These studies were conducted in buffer
C supplemented with 5 mM G/M with 3.4 mM ATP. The SR
protein-to-mitochondria Cyta ratio was 0.5. Preincubation
with thapsigargin (25.0 µM) reduced Ca2+ activation of
respiration by 88 ± 0.4%. Pretreatment with thapsigargin also
increased the effects of Ca2+ on [NADH] (75 ± 0.7%
over baseline; n = 4) compared with control (7.0 ± 0.1% over baseline; n = 4) (see Fig.
6). The addition of ADP (200 µM) is
also shown to illustrate the maximum rate of respiration and the
classic oxidation of NADH in the presence of thapsigargin. Thapsigargin
had no effect on the rate of ADP-stimulated respiration. These data are
consistent with the SR Ca2+-ATPase being responsible for
the majority of the Ca2+-dependent ATPase activity in this
preparation. The increase in [NADH] with Ca2+ in the
presence of Ca2+-ATPase inhibition is consistent with the
activation of CaDH without the balanced increase in ATP
hydrolysis/production that occurs under these conditions.
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Effect of SR-to-mitochondria ratio.
The purpose of this study was to characterize the net effects of
steady-state changes in [Ca2+] on a mixture of porcine SR
vesicles and mitochondria. Clearly, any net effects will depend on the
ratio of SR to mitochondria. We evaluated the effect of the
SR-to-mitochondria content ratio on the respiratory rate and NADH
transition induced by Ca2+ additions in an SR-mitochondria
preparation outlined above. The oxygen consumption rate is expressed as
the change in respiration induced by the addition of 602 nM
Ca2+ (Fig. 7A).
[NADH] is expressed as the change in NADH occurring after the
addition of 602 nM Ca2+ in the presence of different
concentrations of SR holding the mitochondria concentration constant at
1 nM Cyta (Fig. 7B). With increasing SR
concentration the addition of Ca2+ resulted in a
progressively smaller increase in [NADH] as the Ca2+-ATPase activity increased, counteracting the
activation of CaDH. A SR (mg protein)-to-Cyta (nmol) ratio
of 0.5:1 resulted in a near balance between ATP synthesis activation
(via CaDH) and ATP hydrolysis based on the near constant [NADH]. At
higher SR-to-mitochondria ratios, the effect of Ca2+
resulted in a net oxidation of NADH, as the ATPase activity dominated. These studies are the basis for the SR-to-mitochondria ratio of 0.5 used in these studies.
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DISCUSSION |
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This study demonstrates that porcine heart SR and mitochondria have similar steady-state kinetics for Ca2+ activation of ATPase and ATP synthesis, respectively. With the use of this property, conditions were generated in a reconstituted SR-mitochondria mixture in which increases in [Ca2+] generated large increases in ATP production with minimal changes in one of the key metabolic intermediates, NADH. This homeostasis of [NADH] occurred over a wide range of [Ca2+] and ATP turnover rates. These data suggest that the similar kinetics of Ca2+ for SR ATPase activity and mitochondrial metabolic activity may be partially responsible for the maintenance of energy metabolism intermediates during changes in cardiac workload.
The steady-state apparent affinity constants for Ca2+ in the SR and mitochondria are similar to values published in the literature. The SR apparent affinity is clearly variable depending on the phosphorylation state of phospholamban (18), with values ranging between ~150 and 660 nM Ca2+, and the current study was midway between these values (27). The nH of 2 for the SR is consistent with the literature and likely relates to the two binding sites on the Ca2+-ATPase (17, 27). The apparent affinity of Ca2+ for activating the mitochondrial CaDH and the overall ATP synthetic activity fit a simple exponential curve with no evidence of cooperativity in the data despite the fact that Ca2+ is believed to operate within several sites in the mitochondrial metabolic system. A more extensive study evaluating much lower free [Ca2+] levels might reveal a complex Ca2+ dependence. Despite these shifts in the shapes of the curves, the steady-state kinetics of the SR ATPase activity and the mitochondrial ATP synthetic activity are similar (see Fig. 1). These results suggest that these two reactions are poised to respond to changes in [Ca2+] in a fashion that is consistent with Ca2+ playing a role as the cytosolic signaling molecule between these two organelles. Naturally, the K1/2 of these reactions is only one of the parameters involved in the activation by Ca2+ because the maximum velocity of reaction (i.e., relative concentration of elements) and the response times of these processes also have to be accounted for in this analysis. These latter issues are discussed below.
In agreement with the steady-state kinetic data, we found that the effects of [Ca2+] in the reconstituted SR-mitochondria system could be balanced between the synthesis and the breakdown of ATP. This was revealed by the ability to maintain mitochondrial [NADH] nearly constant over a wide range of [Ca2+] and associated ATP synthetic rates driven by the SR Ca2+-ATPase. This is in sharp contrast to a simple increase in ATPase activity alone that is associated with a large decrease in [NADH] (Figs. 2 and 5). The slight increase in [NADH] with [Ca2+] in the SR-mitochondria system might reflect the higher affinity of CaDH for [Ca2+] than the SR ATPase (see Fig. 1) in this in vitro system. In this study, the SR-mitochondria preparation was capable of increasing the ATP production rate nearly fivefold in response to Ca2+ but with only minimal changes in NADH levels (Fig. 5). Thus the flux through NADH had increased by many fold with little change in the concentration of the NADH metabolite. This phenomenon is very similar to that observed in the intact heart (15, 39) during physiological work transitions and suggests that matching Ca2+ sensitivity of the SR and mitochondria metabolic processes may play a role in this process. The current study only examined the NADH levels and not the other intermediates (such as ATP, ADP, Pi, CrP, and Cr) or the mitochondrial membrane potential. Previous studies using non-Ca2+-sensitive ATPase with Ca2+ demonstrated that NADH is an excellent marker of the ATP, ADP, and Pi levels within this system (34), and therefore it is reasonable to assume that the NADH level is an acceptable surrogate for following the metabolic intermediate responses of this system.
The steady-state Ca2+ dependence of SR or mitochondria activity is useful in evaluating the potential interactions of these two pathways in the cell; however, there are important temporal aspects of Ca2+ signaling in the myocyte that must be considered. Myocyte Ca2+ levels change with a complex waveform and with a fundamental frequency similar to the heart rate. The waveform of the cytosolic [Ca2+] suggests that the time dependence of these two systems on Ca2+ may be more important than the steady-state behavior determined herein. That is, although these steady-state kinetic values are useful at one level, they must be convolved with an impulse response of this system to simulate how they will vary according to transient changes in cytosolic [Ca2+]. It is reasonable to assume that the SR must respond to Ca2+ on the scale of the temporal changes in cytosolic [Ca2+], because this pump is actually responsible for the removal of Ca2+ from the cytosol. The temporal response of mitochondria to Ca2+ is more controversial. Earlier studies suggested that the rate of Ca2+ transport into mitochondria is much too slow to respond to the transient changes in cytosolic [Ca2+] that occur during cardiac contraction (5, 6). This implies that the mitochondria will integrate, over some slower time scale (seconds), the Ca2+ levels within the cell and will not respond to rapid transients that occur on a beat-to-beat basis. However, recent studies suggested that the mitochondria metabolic response time to Ca2+ is on the order of 100 ms (35), and rapid uptake mechanisms have also been described (12). The current study does not have the temporal resolution to evaluate the response time of mitochondria on the level of the cardiac cycle. The precise determination of the kinetics of the Ca2+ response awaits sampling schemes more rapid than those used in the current study. Furthermore, the metabolic pool sizes, especially with regard to the "cytosol space" vs. SR and mitochondrial volumes in this highly diluted sample, are seriously distorted in this in vitro preparation. For example, the rate of change of the entire chamber ADP concentration during SR ATPase activation would be predictably much slower than in the cell, where the SR makes up >7% of the volume, in contrast to the fraction of a percent as used in these studies. Clearly, these types of kinetic studies must be conducted not only with faster instrumentation but also with more appropriate reaction conditions that replicate intracellular pool sizes. It is also important to note that permeabilized cells do not resolve this latter issue because the entire incubation medium becomes the cytosol, again distorting the metabolite pool sizes.
This study focused on the interaction of the SR Ca2+-ATPase
with mitochondria. The SR contains less ATPase activity than the contractile apparatus of the heart. We used the SR preparation because
it was much easier to generate and control than muscle filaments in
vitro. In addition, the in vitro or skinned cardiac muscle preparations
have much different Ca2+ dependence of contraction than the
intact muscle. Thus the in vitro contractile apparatus models may not
accurately reflect the in vivo Ca2+ dependence. For these
reasons, we choose to only compare literature data on the
Ca2+ dependence of muscle force in situ with our in vitro
SR and mitochondria data. The Ca2+ dependence of the myosin
ATPase activity has been determined in the steady state by several
investigators [for example, see Backx et al. (2)]. The
steady-state [Ca2+] dependencies for force generation in
the rabbit heart along with the model fits of the data for porcine SR
and mitochondria are presented in Fig. 8.
In general, the Ca2+ dependence for contractile force is
much steeper (i.e., higher apparently cooperative
nH of ~5) and with a much higher midpoint [Ca2+] (650 nM) than determined for SR and mitochondria.
This higher affinity of the SR for Ca2+ makes sense because
the SR must remove Ca2+ below the sensitivity of the
myofilaments to return the heart to diastole [Ca2+]. The
even higher sensitivity of the mitochondria to Ca2+
suggests that the mitochondrial metabolic state could be regulated by
diastolic [Ca2+] or "mean" (i.e., time averaged)
cytosolic [Ca2+]. Another possibility is that the higher
mitochondrial steady-state affinity for Ca2+ may compensate
for a slower response time to the Ca2+ transients,
resulting in a significant metabolic response to even a single
contraction cycle. This latter possibility has not been experimentally
evaluated. In any event, the estimated Ca2+ dependencies of
these different processes involved in the energy metabolism of the
heart are consistent with Ca2+ playing a role in
communication and orchestrating a balanced activation of these two
processes.
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As discussed above, the concentration ratio of SR and mitochondria is critical in this analysis as experimentally verified (see Fig. 7). The SR volume fraction has been estimated to be from 2 to 7% of the cytosolic volume, whereas the mitochondrial volume fraction is approximately ~20% (9, 32). Taking into account the large surface-to-volume ratio of the SR relative to the mitochondria, the ratio of SR and mitochondrial protein might be significantly higher than the volume ratio of these organelles. We determined that a SR-to-mitochondria ratio of 0.5:1 provided a balanced activation of metabolism and ATPase turnover in this steady-state in vitro system. In the cell, the SR will be operating closer to Vmax conditions immediately after the release of Ca2+ into the cytosol, because of the higher cytosolic [Ca2+] (~1 µM) and lower SR [Ca2+] realized under these activated conditions. The Vmax for SR ATP hydrolysis was estimated with a Ca2+ ionophore and was two times higher than the control conditions (see MATERIALS AND METHODS). Thus, if the SR was operating near the Vmax ATP hydrolysis rate, the effective SR-to-mitochondria protein ratio would be closer to 0.25:1 to achieve the balanced activation. This is much closer to the cellular ratio of these two organelles in heart cells. Naturally, this in vitro preparation also eliminates any spatial colocalization that could occur between these organelles in vivo. However, if these systems are communicating via the [Ca2+] even in small gaps, the general concentration dependencies should still be reflected in these more global studies.
Several limitations of the current study design have been discussed above, but others should be mentioned. For example, the use of isolated mitochondria or SR is problematic because modifications can occur during the isolation. This includes changes in affinities for Ca2+ as well as in the velocities of the reactions and modification of carbon substrate sources. The depletion of Ca2+ in the mitochondria is also problematic because it is unclear what the resting matrix Ca2+ is in vivo. Therefore, it is difficult to know whether our protocol mimics the intact heart mitochondria matrix condition. Ca2+ transport can uncouple and overload mitochondria, resulting in membrane permeability changes and irreversible damage. We showed previously (35) that if the steady-state free [Ca2+] in this preparation remains in the range of ~0-600 nM, no uncoupling of respiration is observed. Higher, steady-state [Ca2+] led to mitochondrial effects consistent with high Ca2+ transport levels and the Ca2+-induced permeability transition. Such toxic effects of Ca2+ were avoided in this study. The fact that these relatively low steady-state [Ca2+] (i.e., >700 nM) are toxic to heart mitochondria is problematic, because peak systolic [Ca2+] reaches twofold higher values. The reason for this discrepancy is unknown but might be related to the transient nature of the [Ca2+] in vivo, an enhanced Ca2+ influx, or inhibited Ca2+ efflux in this in vitro preparation.
In summary, the steady-state kinetic dependence of cardiac SR ATP hydrolysis and of mitochondrial ATP production on [Ca2+] are similar. In a reconstituted SR-mitochondria system, a wide range of [Ca2+] resulted in an approximately fivefold change in ATP and NADH turnover with only small changes in mitochondrial [NADH]. These data are consistent with the notion that cytosolic Ca2+ plays a role in the orchestration of energy metabolism in cardiac cells. Cytosolic Ca2+ can modulate both ATP hydrolysis and synthesis in a way that may contribute to the homeostasis of energy metabolism intermediates during alterations in cardiac workload.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. S. Balaban, Laboratory of Cardiac Energetics, National Heart Lung and Blood Institute, National Institutes of Health, Bldg. 10, Rm. B1D-416, Bethesda, MD 20892-1061 (E-mail: rsb{at}nih.gov).
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.
10.1152/ajpcell.00129.2002
Received 20 March 2002; accepted in final form 23 August 2002.
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