Reduced Ca2+ uptake by mitochondria in pyruvate dehydrogenase-deficient human diploid fibroblasts

Rodolfo A. Padua, Kyle T. Baron, Bhaskar Thyagarajan, Colin Campbell, and Stanley A. Thayer

Department of Pharmacology, University of Minnesota Medical School, Minneapolis, Minnesota 55455

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

Physiological and pathological Ca2+ loads are thought to be taken up by mitochondria via a process dependent on aerobic metabolism. We sought to determine whether human diploid fibroblasts from a patient with an inherited defect in pyruvate dehydrogenase (PDH) exhibit a decreased ability to sequester cytosolic Ca2+ into mitochondria. Mobilization of Ca2+ stores with bradykinin (BK) increased the cytosolic Ca2+ concentration ([Ca2+]c) to comparable levels in control and PDH-deficient fibroblasts. In normal fibroblasts transfected with plasmid DNA encoding mitochondrion-targeted apoaequorin, BK elicited an increase in Ca2+-dependent aequorin luminescence corresponding to an increase in the mitochondrial Ca2+ concentration ([Ca2+]mt) of 2.0 ± 0.2 µM. The mitochondrial uncoupling agent carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone blocked the BK-induced [Ca2+]mt increase, although it did not affect the [Ca2+]c transient. Basal [Ca2+]c and [Ca2+]mt in control and PDH-deficient cells were similar. However, confocal imaging of the potential-sensitive dye JC-1 indicated that the percentage of highly polarized mitochondria was reduced from 30 ± 1% in normal cells to 19 ± 2% in the PDH-deficient fibroblasts. BK-elicited [Ca2+]mt transients in PDH-deficient cells were reduced to 4% of control, indicating that PDH-deficient mitochondria have a decreased ability to take up cytosolic Ca2+. Thus cells with compromised aerobic metabolism have a reduced capacity to sequester Ca2+.

intracellular calcium concentration; aerobic metabolism; apoaequorin gene; D-myo-inositol 1,4,5-trisphosphate-sensitive calcium stores; inherited metabolic disorder

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

TRANSPORT OF Ca2+ across the inner mitochondrial membrane is governed by several pathways. Ca2+ entry into the mitochondrial matrix is mediated by a ruthenium red-sensitive electrophoretic uniporter driven by the respiration-dependent proton electrochemical gradient, whereas Ca2+ extrusion into the cytoplasm occurs through Na+-dependent and -independent Ca2+ exchangers (11, 27). Mitochondria take up Ca2+ in response to both physiological and pathological Ca2+ loads. Physiological cytosolic Ca2+ concentration ([Ca2+]c) increases resulting from Ca2+ influx across the plasmalemma or Ca2+ release from intracellular compartments are taken up by mitochondria (9, 11, 15, 23, 33, 34, 41, 47). The activities of Ca2+-sensitive dehydrogenases and other Ca2+-sensitive metabolic processes in the matrix couple ATP production to cellular energy needs (12, 24). For large Ca2+ loads, mitochondrial Ca2+ uptake can act as a buffer to alter the amplitude and shape of the [Ca2+]c response (9, 17, 47). Under pathological conditions, the mitochondrial Ca2+-buffering system can rapidly sequester excessively large Ca2+ loads and thus may protect cells from damage resulting from overactivity of Ca2+-sensitive processes in the cytoplasm (13, 28, 39). Alternatively, mitochondrial Ca2+ uptake may result in cell toxicity from Ca2+-induced uncoupling of oxidative phosphorylation leading to ATP depletion and free radical generation (2, 8, 45, 46, 48).

Patients afflicted with mitochondrial disorders exhibit neurological deficits (44). Although ATP depletion has been suggested to be the primary cause of cellular degeneration, it is currently unclear whether other mechanisms may be involved. Indeed, Moudy et al. (26) have shown that fibroblasts from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) syndrome exhibit elevated [Ca2+]c and a decreased mitochondrial membrane potential. Because mitochondrial Ca2+ sequestration is dependent on the proton electrochemical gradient, we hypothesized that hereditary defects in mitochondrial proteins that decrease aerobic metabolism would result in reduced mitochondrial Ca2+ sequestration. We tested this hypothesis by direct measurement of mitochondrial Ca2+ concentration ([Ca2+]mt) in fibroblasts taken from a patient with reduced pyruvate dehydrogenase (PDH) activity. [Ca2+]mt was determined using a method developed by Rizzuto et al. (33, 34), in which cells were transfected with a cDNA encoding the mitochondrial targeting sequence of cytochrome c fused to the gene for the Ca2+-sensitive photoprotein apoaequorin.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell culture. Control (normal levels of PDH activity; passages 7-15) and PDH-deficient (30% of control PDH activity; passages 2-10) human diploid fibroblasts were a gift from Dr. Robert O'Dea (Departments of Pediatrics and Pharmacology, University of Minnesota, Minneapolis, MN). Cells were seeded at a density of 1 × 106 cells/10-cm dish or 1.7 × 105 cells/25-mm glass coverslip and grown to confluence in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum in a humidified atmosphere of 95% air-5% CO2 at 37°C.

Transfections. The calcium phosphate technique was used to transfect control and PDH-deficient fibroblasts (20). Supercoiled pMT2/mtAEQ (20 µg; Molecular Probes, Eugene, OR) and supercoiled pRSVedl884 (5 µg) (31) were added to cells grown to confluence on 25-mm glass coverslips. Cells were taken for experimentation 48 h following transfection. The presence of the simian virus 40 (SV40) T antigen allowed the pMT2/mtAEQ plasmid to replicate to high copy number, thereby greatly enhancing intramitochondrial apoaequorin protein levels in cotransfected cells. Quantification of intracellular plasmid was performed as previously described (43).

Aequorin measurements. The luminescence detection system employed here (Fig. 1) was custom built and was derived from that described previously by Cobbold and Lee (3). Light emission from aequorin was detected with a low-noise photomultiplier and discriminator/amplifier (Thorn EMI). The output was sampled at 1 Hz with a Thorn EMI CT1 photon-counting board installed in a microcomputer. Apoaequorin was reconstituted in situ by incubation of the transfected cells for 4-8 h with 5 µM coelentrazine in serum-free DMEM at 37°C before each experiment. After reconstitution, the coverslip with cells was mounted in the perfusion chamber, placed in the luminometer, and raised immediately to within 10 mm of the photocathode. Excess coelentrazine was washed away by superfusion of cells for 5 min before recording. At the end of each experiment, unconsumed aequorin was discharged by lysing in H2O that contained 12.6 mM CaCl2. Total aequorin light emission was determined by integration of light emitted during the entire recording.


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Fig. 1.   Flow-through luminometer for adherent cells, used to measure light emission from cultures expressing mitochondrion-targeted recombinant aequorin; 25-mm coverslips with apoaequorin-transfected cells attached were placed in a stainless steel perfusion chamber that was raised close to photocathode. Chamber sits in a base plate that is pressed against shutter housing to provide a light-tight seal. Photons detected by photomultiplier tube were quantified by a photon-counting board in a microcomputer. A: perspective drawing showing luminometer in loading position with shutter closed. B: cross-sectional drawing of luminometer in operating configuration with shutter open. Schematics in A and B are labeled as follows: a, photomultiplier tube housing; b, shutter housing; c, photocathode; d, buffer inlet; e, buffer outlet; f, recording chamber; g, coverslip-retaining plate; h, base plate; i, chamber elevator; j, guide; k, leg; l, movable base plate support. C: recombinant apoaequorin was reconstituted with coelentrazine and challenged with Ca2+ standards in 1 mM Mg2+ at room temperature. For 1 µM < Ca2+ concentration ([Ca2+]) < 5 µM, data were fit with a linear regression of slope 3.8 and y-intercept 18. Slope is in reasonable agreement with 3 Ca2+ binding sites on aequorin (3).

Calibration of aequorin luminescence. Ca2+ binding to aequorin increases the discharge rate such that the log rate of aequorin decay is linearly related to log [Ca2+] (3). We generated a calibration curve (Fig. 1C) at room temperature in buffer that contained (in mM) 20 N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES; pH 7.4), 126 KCl, 10 NaCl, 1 MgCl2, and the indicated concentrations of Ca2+ buffered with ethylene glycol-bis(beta -aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA). Log alpha , where alpha  is photon counts per second divided by remaining counts, was proportional to log [Ca2+] for 1 µM < [Ca2+] < 5 µM. Luminescence signals were converted to [Ca2+]mt by the equation [Ca2+] = 100.26(log alpha  - 18), which describes the linear portion of the curve (38). Because the rate of aequorin discharge is low for [Ca2+] <1 µM this method lacks precision for measuring small changes in basal [Ca2+]mt. The ionic composition of the matrix is not known, particularly the [Mg2+], which is the ion concentration with the most significant influence on aequorin luminescence. Thus the calibration curve might be shifted if our estimation of matrix [Mg2+] were inaccurate.

Photometry and digital imaging. The instrumentation and calibration for indo 1-based dual-emission microfluorometry and fura 2-based digital imaging have been described previously (19). Fibroblasts were incubated for 45-60 min at 37°C in either 2 µM indo 1-acetoxymethyl ester (AM), for photometry, or 2 µM fura 2-AM, for digital imaging, in HEPES-buffered Hanks' salt solution that contained 0.5% bovine serum albumin. Hanks' buffer had a composition (in mM) of 20 HEPES, 137 NaCl, 1.3 CaCl2, 0.4 MgSO4, 0.5 MgCl2, 5.0 KCl, 0.4 KH2PO4, 0.6 Na2HPO4, 3.0 NaHCO3, and 5.6 glucose. Loading was terminated by washing the coverslip three times with Hanks' buffer before the start of an experiment. A coverslip with cells was then mounted in a flow-through chamber (42), placed on the stage of the microfluorometer, and superfused at a rate of 2 ml/min. The flow characteristics of the chambers used in luminescence and microfluorometry experiments were identical. Solutions were selected with a multiport valve coupled to several reservoirs. CaCl2 was replaced with 1 mM EGTA for Ca2+-free experiments.

Measuring mitochondrial membrane potential. Mitochondrial membrane potential was determined essentially as described by Moudy et al. (26). The fluorescent indicator JC-1 is taken up by mitochondria as a monomer (522-nm emission) that when concentrated in highly polarized mitochondria forms J-aggregates (605-nm emission). Thus, when excited at 488 nm, the ratio of the emission intensities at 605 and 522 nm can be used as an index of mitochondrial membrane potential. Fibroblasts were incubated in 0.5 µM JC-1 for 10 min and imaged with a Bio-Rad MRC 1024 laser scanning confocal microscopy system mounted on an Olympus AX70 microscope with a ×60 (numerical aperture = 0.9) water immersion objective. The dye was excited at 488 nm and emitted light directed with a 560-nm dichroic mirror through 605-nm (32) and 522-nm (32) band-pass filters to photomultiplier tubes. Ratio values were calculated from pixels that were within a mask defined by the 522-nm image.

Statistical analyses. Data are presented as means ± SE, and significance was determined with Student's t-test. Each transfection of six coverslips was designated as one experiment (n = 1).

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

In human diploid fibroblasts, increases in [Ca2+]c were elicited by superfusion of cells with 100 nM bradykinin (BK). BK has been previously shown to evoke the release of Ca2+ stored in D-myo-inositol 1,4,5-trisphosphate (IP3)-sensitive intracellular Ca2+ stores in these cells (10). As shown in Fig. 2A, 60-s application of BK elicited a rapid and transient increase in [Ca2+]c in control fibroblasts. [Ca2+]c rose from a basal level of 83 ± 12 nM to peak at 1,131 ± 151 nM (n = 7). A second exposure to BK elicited a second [Ca2+]c transient with a peak [Ca2+]c of 777 ± 203 nM (Fig. 2A). In digital [Ca2+]c-imaging experiments, 89% of the cells in a chosen field exhibited at least three [Ca2+]c transients following repeated exposures to BK (n = 9 cells in 2 coverslips). Thus indo 1-based photometry experiments such as those shown in Fig. 2, A and D, are accurate representations of the field as a whole. To determine whether BK-induced increases in [Ca2+]c were sequestered by mitochondria, fibroblasts were transfected with the gene encoding mitochondrion-targeted apoaequorin (mtAEQ). In cells transfected with mtAEQ, BK elicited an increase in [Ca2+]mt, as indicated by a marked increase in Ca2+-dependent aequorin light emission (Fig. 2B). Cells were lysed in the presence of high Ca2+ at the end of each experiment. Light emitted during the entire recording was summed (3.6 ± 0.5 × 105 photon counts; n = 23) and total photon counts were used to calculate the rate of aequorin decay, a value that is proportional to [Ca2+] and independent of both transfection efficiency and the consumption of aequorin (3). Thus, as shown in Fig. 2C, even though the second BK application elicited a smaller change in luminescence intensity relative to the first, when luminescence signal was converted to [Ca2+], the responses were shown to be of similar amplitude (peak 2/peak 1 = 0.73 ± 0.03). Basal [Ca2+]mt was 1.9 ± 0.1 µM, and the net change in [Ca2+]mt following BK exposure was 2.0 ± 0.2 µM (n = 7). If the calibration of the indo 1-based and mtAEQ-based methods are assumed to be comparable, these data indicate that during a [Ca2+]c transient the matrix [Ca2+] is much higher, suggesting that mitochondria use the membrane potential across the inner membrane to concentrate Ca2+. The rise in [Ca2+]c induced by BK was mainly due to release of Ca2+ from intracellular compartments, because removal of extracellular Ca2+ did not significantly affect the amplitude (peak 1/peak 2 = 1.0 ± 0.2; n = 3) of the BK-elicited [Ca2+]c transient (Fig. 2D).


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Fig. 2.   Bradykinin (BK)-induced Ca2+ mobilization increases cytosolic [Ca2+] ([Ca2+]c) and mitochondrial [Ca2+] ([Ca2+]mt) in human diploid fibroblasts. BK (100 nM), 12.6 mM Ca2+-H2O (Ca2+/H2O) and Ca2+-free 1 mM EGTA (0 Ca2+) were applied at times indicated by horizontal bars. A: BK evoked an increase in [Ca2+]c that was reproducible. B: luminescence from mitochondrion-targeted apoaequorin (mtAEQ)-transfected fibroblasts exposed to BK. Total luminescence was estimated at end of each experiment by lysing cells with hyposmotic medium (Ca2+/H2O) to discharge unconsumed aequorin. cps, Counts/s. C: [Ca2+]mt values calculated from B show that, although luminescence of 2nd BK exposure was of much lower intensity relative to 1st, BK-elicited increase in [Ca2+]mt was of similar amplitude following conversion of luminescence to [Ca2+]mt. D: BK-elicited [Ca2+]c transients are mediated by release of Ca2+ from intracellular stores (n = 3).

To determine the extent to which Ca2+ uptake into the mitochondria shapes the BK-induced [Ca2+]c transient, fibroblasts were treated with the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), a protonophore that dissipates the electrochemical gradient across the inner mitochondrial membrane, thus disrupting the ability of mitochondria to sequester Ca2+ (Fig. 3A). FCCP alone elicited a small [Ca2+]c transient. FCCP did not significantly affect BK-induced changes in [Ca2+]c (Fig. 3D; n = 4), suggesting that mitochondria do not significantly contribute to [Ca2+]c buffering in fibroblasts following exposure to BK. Thus, in human diploid fibroblasts, mitochondria take up Ca2+ during BK-induced increases in [Ca2+]c, as indicated in Fig. 2, B and C, by BK-induced aequorin luminescence, but the amount of Ca2+ sequestered does not appear to be large enough to significantly alter the shape of the [Ca2+]c transient. To confirm that the source of this aequorin luminescence was the mitochondrion, FCCP was applied to mtAEQ-transfected cells before and during BK exposure (Fig. 3, B and C). FCCP effectively blocked the BK-induced luminescence increase (Fig. 3D). This response was restored on removal of the uncoupler (Fig. 3, B and C). Thus, in mtAEQ-transfected fibroblasts, BK evoked luminescence responses that resulted from an increase in [Ca2+]mt. Because FCCP did not affect the shape of the BK-induced [Ca2+]c transient, the amount of Ca2+ in the cytosol taken up by the mitochondria must be small relative to the total Ca2+ released from internal Ca2+ stores following a BK stimulus.


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Fig. 3.   Uncoupling aerobic metabolism inhibits Ca2+ uptake into mitochondria. BK (100 nM), 12.6 mM Ca2+-H2O (Ca2+/H2O), and FCCP (1 µM) were applied at times indicated by horizontal bars. A: addition of FCCP during exposure to BK did not alter shape of BK-elicited [Ca2+]c transient (n = 3). B and C: FCCP blocked BK-elicited increase in luminescence (B) and [Ca2+]mt (C). They were restored on removal of uncoupler. D: histogram summarizes effects of FCCP on [Ca2+]c and [Ca2+]mt. * P < 0.0001 vs. mitochondrial control.

Clearly, uncoupled mitochondria fail to take up Ca2+. We next sought to determine whether cells in which aerobic metabolism was more subtly compromised might also exhibit reduced mitochondrial Ca2+ uptake. We obtained fibroblasts from a patient with an inherited defect in the E1 component of the PDH complex that resulted in activity levels ~30% of normal. Because PDH is a rate-limiting enzyme in the metabolism of carbohydrates, reduction in PDH activity has been associated with an assortment of neurological and metabolic deficits (44). In culture, the PDH-deficient fibroblasts replicated far more slowly than normal cells and had a flat, more spread-out appearance. Presumably the reduction in PDH activity produces a depolarization of the mitochondrial membrane potential.

We tested this hypothesis by imaging normal and PDH-deficient fibroblasts loaded with the fluorescent dye JC-1. This dye is taken up by mitochondria as a monomer (522-nm emission) that when concentrated in highly polarized mitochondria forms J-aggregates (605-nm emission). In Fig. 4A, confocal images of normal fibroblasts loaded with JC-1 are shown. Frame 1 shows the fluorescence emitted by the aggregates, frame 2 shows the monomers, and in frame 3 the two images are overlaid. Note that the aggregate image is a subset of the monomer image. Thus the monomer image was used as a mask to define mitochondria, and then frame 1 was divided by frame 2 to produce the ratio images shown in frame 4. The same images collected from PDH-deficient cells (Fig. 4B) showed similar images of the monomer but had a reduced number of J-aggregates. This is apparent when the data from seven normal and six PDH-deficient cells are presented as frequency distributions. Note the decreased number of pixels with ratios >2.5, indicative of highly polarized mitochondria, and the increased number of pixels with ratios <1 in the PDH-deficient cells. As shown in the inset of Fig. 4, the percentage of pixels >1.25, indicative of polarized mitochondria, was significantly greater in normal (30 ± 1%) relative to PDH-deficient cells (19 ± 2%, P < 0.005). This frequency distribution is in excellent agreement with that described for MELAS fibroblasts, which also displayed a reduction in the number of highly polarized mitochondria (26).


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Fig. 4.   Mitochondrial membrane potential is reduced in pyruvate dehydrogenase (PDH)-deficient fibroblasts. Normal (A) and PDH-deficient (B) fibroblasts were loaded with JC-1 and imaged with confocal microscopy as described in EXPERIMENTAL PROCEDURES. Frame 1 shows image collected at 605 nm to detect J aggregates indicative of highly polarized mitochondria. Frame 2 shows image collected at 522 nm to detect JC-1 monomers that identify all mitochondria. In frame 3, frames 1 and 2 are overlaid. Frame 4 shows pseudocolor representations of ratio values that result from dividing frame 1 by frame 2. Pixels were considered mitochondrial if they resided within a mask generated from 522-nm image (frame 2). C: histogram displays frequency distribution of aggregate-to-monomer ratio values for 7 normal and 6 PDH-deficient cells. Inset compares average percentage of pixels with ratio values <1.25 in normal (n = 7 cells) and PDH-deficient cells (n = 6 cells). * P < 0.005 vs. normal.

In PDH-deficient cells, BK elicited increases in [Ca2+]c (peak amplitude of 885 ± 74 nM, n = 7; Fig. 5, A and E), that were not significantly different from those recorded from control fibroblasts. Multiple exposures to BK elicited reproducible [Ca2+]c transients similar to those in control fibroblasts (Fig. 5A). Thus the PDH deficiency in these cells did not affect their ability to mobilize and refill IP3-sensitive Ca2+ stores. Presumably, as is true for most cells maintained in culture, the fibroblasts relied primarily on glycolysis for energy production (22). As shown in Fig. 5B, FCCP did not significantly affect the change in the [Ca2+]c induced by BK (n = 3).


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Fig. 5.   BK-induced [Ca2+]c responses in PDH-deficient fibroblasts are normal, but [Ca2+]mt transients are reduced. BK (100 nM) and 12.6 mM Ca2+-H2O (Ca2+/H2O) were applied at times indicated by horizontal bars. A: BK elicited [Ca2+]c transients that were reproducible (n = 4). B: FCCP did not affect shape of BK-elicited [Ca2+]c transient (n = 3). C and D: stimulation with BK failed to elicit an increase in luminescence (C) or [Ca2+]mt (D) in PDH-deficient fibroblasts. mtAEQ was expressed as indicated by luminescence and [Ca2+]mt transients elicited by hyposmotic lysis (Ca2+/H2O). E: comparison of BK-elicited increases in [Ca2+]mt in control and PDH-deficient fibroblasts indicates a dramatic reduction in sequestration of BK-induced [Ca2+]c increase by mitochondria in PDH-deficient cells. * P < 0.0001 vs. mitochondrial control.

Although BK-induced [Ca2+]c transients appeared normal in PDH-deficient cells, [Ca2+]mt increases in these cells were greatly reduced relative to cells from a normal donor. In PDH-deficient fibroblasts transfected with mtAEQ, BK elicited very small, often undetectable [Ca2+]mt increases (0.07 ± 0.03 µM, n = 4; Fig. 5, C and D). In some cells that failed to respond to BK, 10 µM ionomycin evoked a modest response (0.2 ± 0.1 µM). Basal [Ca2+]mt (2.5 ± 0.1 µM) was not significantly different from that of control cells. Coverslips that lacked a detectable luminescence response to hyposmotic lysis in 12.6 mM Ca2+ were excluded from the data set (12 of 17 coverslips) because [Ca2+]mt cannot be measured in cells lacking aequorin. However, even PDH-deficient cells in which a significant level of luminescence was detected, such as those used to generate the recording in Fig. 5C, failed to display a [Ca2+]mt increase in response to BK. Total luminescence recorded from PDH-deficient cells was 27% of that recorded from normal cells, suggesting a lower level of apoaequorin expression in PDH-deficient mitochondria relative to normal mitochondria. Control experiments indicated that both wild-type and PDH-deficient cells contained roughly equivalent amounts of transfected plasmid DNA (data not shown). Note that the conversion of luminescence to [Ca2+] depends on the rate of aequorin consumption and not on the total amount present. Thus, despite the lower mtAEQ expression in PDH-deficient cells, there was no correlation between the amplitude of BK-elicited [Ca2+]mt transients and total photon counts in either PDH-deficient or control fibroblasts. Clearly, mitochondria in PDH-deficient fibroblasts have a reduced capacity to sequester cytosolic Ca2+ following BK stimulation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

Direct evidence for parallel increases in [Ca2+]mt and [Ca2+]c in intact cells following agonist stimulation has only recently been forthcoming (34, 35, 38). Here, we utilized an approach developed by Rizzuto and colleagues (35) whereby recombinant apoaequorin targeted to mitochondria (mtAEQ) was transiently expressed at high levels in human diploid fibroblasts. This strategy enabled the direct measurement of [Ca2+]mt following mobilization of Ca2+ stores with BK. We found that, in normal fibroblasts, BK-induced [Ca2+]c transients were paralleled by [Ca2+]mt increases. In contrast, mitochondria in PDH-deficient fibroblasts failed to sequester BK-elicited [Ca2+]c increases, although mtAEQ was still expressed in these cells. Presumably, the decreased Ca2+ uptake resulted from the reduced number of polarized mitochondria in the PDH-deficient cells. These results demonstrate that genetic defects in mitochondrial function impair the sequestration of cytosolic Ca2+ loads and suggest that this inability to take up Ca2+ may contribute to the pathology seen in patients afflicted with metabolic disorders.

In human diploid fibroblasts, BK elicited a [Ca2+]c rise through mobilization of Ca2+ from intracellular stores. This resulted in a large [Ca2+]mt increase consistent with that observed in other cell types following stimulation with specific agonists (34, 35, 38). Application of FCCP during BK treatment, which prevents Ca2+ accumulation by mitochondria, blocked BK-induced light emission in mtAEQ-transfected fibroblasts. This is in agreement with previous results in which FCCP significantly reduced the [Ca2+]mt increase evoked by agonists in endothelial cells (35). Thus FCCP is a useful tool for the rapid and efficacious disruption of mitochondrial Ca2+ uptake at concentrations that leave IP3-sensitive Ca2+ stores intact and functional. This observation suggests that glycolysis can maintain cellular ATP levels sufficient for maintaining the phosphoinositide signaling pathway. Clearly, intact aerobic metabolism is required for Ca2+ uptake by mitochondria but is not needed for the function of intracellular Ca2+ stores. The inability of FCCP to alter the BK-induced increase in [Ca2+]c also highlights the varying degree to which the mitochondria serve to shape changes in [Ca2+]c. The role of the mitochondrion in Ca2+ buffering varies with cell type, stimulus strength, and the source of the Ca2+ load. In excitable cells, the contribution of mitochondria to buffering [Ca2+]c increases with the size of the Ca2+ load (7, 17, 47). The contribution of Ca2+ entering the cytoplasm from various sources to changes measured in the mitochondrial matrix varies with the specific intracellular localization of mitochondria relative to the source of the Ca2+ (21). It is clear from these studies that in all cell types tested mitochondria sequester Ca2+. However, the degree to which this Ca2+ uptake influences changes in [Ca2+]c appears to vary.

A deficiency in PDH is one of the most common defined genetic defects of mitochondrial energy metabolism (18, 32), and, because PDH catalyzes the conversion of pyruvate to acetyl CoA, it is a key regulatory enzyme in carbohydrate metabolism. In some tissues such as brain where energy is derived predominantly from aerobic metabolism, the enzyme operates at near maximal rate (4). Thus relatively modest reductions in PDH activity lead to accumulation of pyruvate and lactic acid, resulting in metabolic and neurological abnormalities (29). In this study, [Ca2+]mt measured in fibroblasts with ~30% of normal PDH activity indicated that Ca2+ uptake activity was reduced to 4% of that recorded in normal cells. Several neurological disorders have been linked to deficiencies of PDH, including Leigh syndrome (30), Huntington's disease (40), Alzheimer's disease (40), and Reye's syndrome (36). It is not clear whether some of the pathology observed in these patients is due to abnormal cellular Ca2+ handling, although disrupted Ca2+ homeostasis and altered mitochondrial function are emerging as important early events in a number of neurological disorders (1). Fibroblasts from patients with MELAS syndrome were shown to have elevated [Ca2+]c and a decreased mitochondrial membrane potential (26). In the PDH-deficient fibroblasts studied here, no significant difference in the resting level of cytosolic Ca2+ was detected, and BK-elicited [Ca2+]c transients were comparable to those seen in normal fibroblasts, suggesting that the PDH deficiency did not affect the ability of cells to release Ca2+ from intracellular stores. However, BK-induced [Ca2+]c increases did not produce [Ca2+]mt transients in PDH-deficient cells as seen in normal fibroblasts. The light emission produced by cell lysis in high-Ca2+ hyposmotic solution indicated that, although aequorin expression was reduced, the PDH deficiency did not prevent its expression altogether.

The reduced number of polarized mitochondria in the PDH-deficient cells presumably accounts for their inability to take up Ca2+. These data suggest that only a relatively small fraction of the total number of mitochondria take up Ca2+ from the cytoplasm, because the principal difference between normal and PDH-deficient cells, which essentially failed to take up Ca2+, was the reduction in the number of highly polarized mitochondria from 30 ± 1 to 19 ± 2%. This observation raises the interesting idea that most of the mitochondria in the cell are not functional but rather serve as a reserve or are in various stages of assembly and degradation. It is intriguing to speculate that treatments might be found to favorably influence this distribution.

Ca2+ accumulation in mitochondria following stimuli that increase [Ca2+]c in the physiological range modulates the activities of the matrix dehydrogenases, including the PDH complex, isocitrate dehydrogenase (NAD+), and the 2-oxoglutarate dehydrogenase complex. All of these enzymes are involved in the catalysis of NADH and are thus potential regulatory sites for oxidative metabolism (25). [Ca2+]mt increases in the low micromolar range can produce a severalfold increase in activity of these enzymes. Moreover, [Ca2+]mt and the activity of Ca2+-dependent mitochondrial dehydrogenases oscillate at the frequency of [Ca2+]c (14, 37), suggesting that the mitochondrion is well suited to act as an integrator of [Ca2+]c, converting signaling information encoded by rapid changes in [Ca2+]c into a time-averaged increase in aerobic metabolism. Interestingly, the [Ca2+]mt following BK-stimulation of fibroblasts appeared to attain levels previously reported to activate matrix dehydrogenases and is consistent with the hypothesis that the transmission of the cytosolic Ca2+ signal to the mitochondrial matrix allows the modulation of mitochondrial metabolism according to cellular energy demands (5, 6, 15, 16, 25).

In summary, we found that an inherited defect in aerobic metabolism impaired mitochondrial Ca2+ uptake, highlighting the direct role of aerobic metabolism in cellular Ca2+ homeostasis. In metabolically compromised cells, two principal effects on Ca2+ signaling are predicted. First, the increased energy demand signaled by increased [Ca2+]c is not detected by rate-limiting Ca2+-sensitive enzymes in the matrix, and, second, the potential for mitochondria to buffer large Ca2+ loads is lost.

    ACKNOWLEDGEMENTS

This work was supported in part by National Science Foundation Grant IBN9723796, National Institute on Drug Abuse Grant DA07304, National Cancer Institute Grant CA61906, and American Heart Association Grant 9601039.

    FOOTNOTES

R. A. Padua is the recipient of a Medical Research Council of Canada Postdoctoral Fellowship.

Address for reprint requests: S. A. Thayer, Dept. of Pharmacology, University of Minnesota Medical School, 3-249 Millard Hall, Minneapolis, MN 55455.

Received 5 June 1997; accepted in final form November 11 1997.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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AJP Cell Physiol 274(3):C615-C622
0363-6143/98 $5.00 Copyright © 1998 the American Physiological Society




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