Role of calcium in metabolic signaling between cardiac sarcoplasmic reticulum and mitochondria in vitro

Robert S. Balaban, Salil Bose, Stephanie A. French, and Paul R. Territo

Laboratory of Cardiac Energetics, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892-1061


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 (Delta Psi ) and the F0/F1-ATPase that utilizes Delta Psi 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.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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 · min-1 · mg-1 (n = 11) at 37°C determined in the presence of the Ca2+ ionophore A-23187 with 2 µM free Ca2+. In the absence of added Ca2+, the SR ATPase rate was 166 ± 15 nmol Pi · min-1 · mg-1 (n = 11). Calcium ionophore was not used in the SR-mitochondria preparations because it would result in mitochondrial Ca2+ overload and subsequent metabolic uncoupling. Thus the passive leak across the SR membrane was used to set the steady-state recycling rate. In the absence of ionophore, the maximum ATPase rate was 972 ± 50 nmol Pi · min-1 · mg-1 (n = 11) at 37°C. Inhibition of Ca2+-ATPase activity with 25 µM thapsigargin (Sigma, St. Louis, MO) resulted in a complete inhibition of the Ca2+-stimulated ATP activity to 120 ± 11 nmol Pi · min-1 · mg-1 (n = 3). These results suggest that the majority of the ATPase activity in this preparation is the Ca2+-sensitive ATPase of the SR.

Cytochrome 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 · min-1 · nmol Cyta-1 (n = 5). Similar percent increases were observed in the presence of G/M with 0.5 g/l SR causing an increase from 39.1 ± 4.0 to 51.1 ± 6.0 nmol O2 · min-1 · nmol Cyta-1 (n = 5). Thapsigargin (25 µM) had no effect on this baseline rate, suggesting that it was due to contamination by non-Ca2+-sensitive ATPase activity. Although this is close to a 70% increase in rate, it is still well below the Ca2+-activated rate of ~250 nmol O2 · min-1 · nmol Cyta-1 in the reconstituted SR-mitochondria system and is consistent with the background ATPase activity in the SR preparation (see Porcine heart SR). No difference in the maximum rate of ATP production stimulated with ADP and Pi was found in the presence or absence of SR vesicles. These results are consistent with no adverse effects of the SR vesicles on isolated mitochondria, including metabolic uncoupling from residual lipids, carbon metabolite sources, or significant non-Ca2+-sensitive ATPase activity. The ratio of mitochondria and SR protein was determined by using the protein content of SR in milligrams vs. nanomoles of Cyta. One nanomole of Cyta is roughly equivalent to 0.9 mg of protein in porcine heart mitochondria (4).

Data 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).


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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-1 · mg protein-1 in the absence of a Ca2+ ionophore. The SR data were fit to a Hill equation of the form
ATPase rate  (1)

= [max rate × ([Ca<SUP>2+</SUP>])<SUP><IT>n</IT><SUB>H</SUB></SUP>]/[<IT>K</IT><SUP><IT>n</IT><SUB>H</SUB></SUP><SUB>1/2</SUB> + ([Ca<SUP>2+</SUP>])<SUP><IT>n</IT><SUB>H</SUB></SUP>]
The best fit occurred with a Hill coefficient (nH) of 1.98 and a half-maximal concentration (K1/2) of 335 nM.


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Fig. 1.   Effect of Ca2+ concentration ([Ca2+]) on apyrase-driven mitochondrial ATP production, state 4 [NADH], and sarcoplasmic reticulum (SR) ATP hydrolysis activity. The [Ca2+] shown is the calculated free concentration. Data are normalized to 100 at maximum [Ca2+] to facilitate comparison. triangle , SR ATP hydrolysis activity; black-lozenge , state 4 [NADH]; , mitochondrial ATP production. Lines are fits of the data as described in RESULTS.

Mitochondrial ATP production data as well as the [NADH] response at state 4 were fit to simple exponential functions because an adequate fit of the data was attained with this simple model. The form of this equation for ATP production was
ATP synthetic rate = <IT>V</IT><SUB>max</SUB> (1 − exp<SUP>−[Ca<SUP>2+</SUP>]/<IT>K</IT></SUP>) (2)
where Vmax is the maximum Ca2+-stimulated rate or the maximum [NADH]. The K fit for apyrase-driven ATP production was 165 nM Ca2+ with the K1/2 calculated to be 114 nM. The K for state 4 [NADH] was 352 nM Ca2+ with a K1/2 calculated to be 243 nM. The model fits were used to provide a mathematical simulation of the data for comparison purposes; no mechanistic significance is placed on these simulations. It is important to note that the K calculated for the state 4 [NADH] data represents the concentration dependence on Ca2+ under near-zero net flux conditions. As such, it represents the influence of Ca2+ on the ratio of NADH production and breakdown under rather nonphysiological conditions.

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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Fig. 2.   Comparison of the effect of [Ca2+] on the SR-mitochondria system with apyrase additions to mitochondria alone. Paired studies from the same day are presented. Dashed line, SR-mitochondria system data; solid line, apyrase system. NADH is expressed as the ratio of the NADH fluorescence signal with the signal at anoxia (100% reduced). Zero [NADH] is taken as the NADH value at the end of the [Ca2+] depletion step. Numbers in brackets on the time line are oxygen consumption rate in nmol O2 · min-1 · nmol cytochrome aa3 (Cyta)-1. The first arrow indicates the addition of 5 mM K-glutamate and K-malate (G/M) to both samples. At the second arrow, 1.5 U/ml of apyrase was added to the chamber, which caused a decrease in [NADH] and an increase in respiration in this system. In the SR-mitochondria system, the second arrow indicates the addition of 300 nM Ca2+, which was associated with a increase in respiration but a slight increase in [NADH]. The mitochondria concentration was 1 µM Cyta in both studies.

The effects of [Ca2+] on the ATP production rate of the SR-mitochondria system are presented in Fig. 3. These experiments were conducted with a SR-to-mitochondria ratio of 0.5 (mg protein/nmol Cyta) and were identical to those in Fig. 2. No fit of these data was performed because [Ca2+] of >500 nM were not used in this study to avoid uncoupling effects (36). This prevented the determination of the maximum Ca2+ activation values in this system (see Fig. 1 for SR ATPase). However, a sigmoidal activation pattern is observed, consistent with the activation of the SR ATPase.


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Fig. 3.   Effect of [Ca2+] on respiratory rate of reconstituted SR and mitochondria system. Free [Ca2+] was calculated as described in MATERIALS AND METHODS. Means ± SE for n = 4 are shown. Mitochondria concentration was 1 µM Cyta. SR concentration was 0.5 g protein/l.

The effects of [apyrase] on ATP turnover rate are presented in Fig. 4. The conditions were identical to those in Fig. 2 for the apyrase conditions. An essentially linear relationship between [apyrase] and ATP turnover was observed. This was expected because the rate of ATP hydrolysis is proportional to [apyrase]. As long as the ATP synthetic capacity of the mitochondria is not exceeded, the turnover of ATP, or oxygen consumption rate, should also be linear with [apyrase].


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Fig. 4.   Effect of apyrase concentration ([apyrase]) on respiratory rate of mitochondrial suspensions. Means ± SE for n = 4 are shown. Mitochondria concentration was 1 µM Cyta.

To illustrate the relationship between [NADH] and ATP turnover under these two conditions the respiratory rate was plotted as a function of [NADH] for experiments where [apyrase] or [Ca2+] was systematically increased (Fig. 5). These data are presented as individual data points and were fit with a simple linear regression. The apyrase data revealed a decrease in [NADH] with increasing respiratory rate, consistent with the classic ADP and Pi activation of oxidative phosphorylation (7). The apyrase experimental data had a slope of -302 nmol O2-1 · min-1 · nmol Cyta-1 · [NADH]-1, a y-intercept of 227 nmol O2-1 · min-1 · nmol Cyta-1, and a x-intercept of 0.85 [NADH]. The SR-mitochondria data demonstrated that [NADH] slightly increased as a function of [Ca2+] and respiratory rate. The Ca2+-activated SR-mitochondria system had a positive slope of 8,046 [NADH] · nmol O2-1 · min-1 · nmol Cyta-1 with an x-intercept of 0.54 [NADH]. It is important to note that the near homeostasis of [NADH] in the SR-mitochondria system occurred over a wide range of [Ca2+] and ATP turnover rates as illustrated in Fig. 3. This implies that this balanced activation created in vitro was not limited to a particular ATP turnover rate or [Ca2+].


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Fig. 5.   Relationship between [NADH] and respiratory rate in the apyrase and SR-mitochondria reconstitution experiments. NADH is normalized as described for Fig. 2. open circle , Varying [apyrase]. Increasing [Apyrase] increased respiration but decreased [NADH]. , Fixed [SR] and [mitochondria] (ratio of SR to mitochondria = 0.5) with varying [Ca2+] from 0 to 492 nM. Increasing [Ca2+] increased respiration with a slight increase in [NADH]. Lines are the linear regression of the data points.

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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Fig. 6.   Effect of thapsigargin on the Ca2+ effect on respiration and [NADH] in the SR-mitochondria reconstituted system. Mitochondria were incubated in buffer C with 5 mM G/M with 3.4 mM ATP. Dashed line, data with 25 µM thapsigargin included in the preincubation medium; solid line, control. Numbers in brackets are the respiratory rate in nmol O2 min-1 nmol Cyta-1. SR and mitochondria concentrations were the same as described for Fig. 2. Initial respiratory rate and [NADH] were identical. The first arrow represents the addition of 492 nM Ca2+ to the chamber. Thapsigargin blocked the respiratory effects of Ca2+ while enhancing the [NADH] response. The second arrow represents the addition of ADP at 200 µM. Thapsigargin had no effect on ADP-driven respiration. Anoxia was reached at different times for the 2 samples, thapsigargin taking longer, because of the difference in the oxygen consumption rate before the addition of Ca2+. SR and mitochondria concentrations were the same as in Fig. 2.

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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Fig. 7.   Effect of SR protein-to-Cyta ratio on metabolic effects of Ca2+ additions. Incubation conditions were identical for the reconstituted SR-mitochondria system as stated in Fig. 2 with the exception that the SR protein-to-Cyta (g/µmol) ratio was varied from 1 to 0. A: effect of SR protein-to-Cyta ratio on the change in respiratory rate. Data are plotted as the SR protein-to-Cyta ratio vs. the change (Delta ) in respiration. Change in respiration was calculated by taking the difference in respiration between the G/M control period and Ca2+-stimulated rate as shown in Fig. 2. B: effect of SR protein-to-Cyta ratio on the change in [NADH]. Again the change in [NADH] was calculated by taking the difference between [NADH] during the glutamate-malate control period and the Ca2+-stimulated level. These results are the mean of 3 replicates conducted on the same population of mitochondria (i.e., conducted on 1 day's preparation).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 8.   Summary of the Ca2+ dependence of mitochondrial ATP production (this study), SR ATP hydrolysis (this study), and cardiac muscle force in situ (Ref. 2). Data are normalized to the maximum Ca2+ effect to ease comparison.

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 284(2):C285-C293