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J. Biol. Chem., Vol. 279, Issue 39, 40328-40336, September 24, 2004
Inhibition of Mitochondrial Na+-Ca2+ Exchange Restores Agonist-induced ATP Production and Ca2+ Handling in Human Complex I Deficiency*![]() ¶||![]() ![]() ¶![]() ![]() ¶![]() ¶|| ¶ || ||¶¶ ¶![]() ![]()
From the
Departments of
Received for publication, July 16, 2004
Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) of the oxidative phosphorylation system is a multiprotein assembly comprising both nuclear and mitochondrially encoded subunits. Deficiency of this complex is associated with numerous clinical syndromes ranging from highly progressive, often early lethal encephalopathies, of which Leigh disease is the most frequent, to neurodegenerative disorders in adult life, including Leber's hereditary optic neuropathy and Parkinson disease. We show here that the cytosolic Ca2+ signal in response to hormonal stimulation with bradykinin was impaired in skin fibroblasts from children between the ages of 0 and 5 years with an isolated complex I deficiency caused by mutations in nuclear encoded structural subunits of the complex. Inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157completely restored the aberrant cytosolic Ca2+ signal. This effect of the inhibitor was paralleled by complete restoration of the bradykinin-induced increases in mitochondrial Ca2+ concentration and ensuing ATP production. Thus, impaired mitochondrial Ca2+ accumulation during agonist stimulation is a major consequence of human complex I deficiency, a finding that may provide the basis for the development of new therapeutic approaches to this disorder.
Human mitochondrial complex I (NADH:ubiquinone oxidoreductase) is the largest multisubunit assembly of the oxidative phosphorylation (OXPHOS)1 system, comprising 39 nuclear encoded and seven mitochondrially encoded subunits. Malfunction of this complex is associated with a wide variety of clinical syndromes ranging from often early lethal disorders, of which Leigh disease, a progressive encephalopathy, is the most frequent, to neurodegenerative disorders in adulthood, including Leber's hereditary optic neuropathy and Parkinson disease. In recent years, all human nuclear structural complex I genes have been characterized, which allowed us to elucidate the genetic defect in 40% of a cohort of complex I-deficient patients in which the enzyme defect was present in at least skeletal muscle and cultured skin fibroblasts (17). To enhance our understanding of the pathophysiological consequences of these diseases, with the final aim of developing new treatment strategies to stabilize or even cure these conditions, we study genetically characterized human complex I-deficient fibroblast cell lines as a model for OXPHOS system disease, knowing that these cells are glycolytic (8). Several hypotheses concerning the pathophysiology of OXPHOS diseases have been investigated of which the most consistent are (a) increased production of reactive oxygen species (9), (b) decreased potential across the mitochondrial inner membrane (10), (c) decreased intracellular ATP levels (1113), and (d) altered Ca2+ homeostasis (10, 12). In agreement with the reactive oxygen species hypothesis (a), we found that metallothioneins were up-regulated in all of the genetically characterized complex I-deficient cell lines (14). Pilot experiments with these cell lines furthermore revealed that human complex I deficiency was associated with altered cytosolic Ca2+ homeostasis. This encouraged us to undertake a detailed analysis of ATP production and calcium handling in a skin fibroblast cell line derived from a Leigh disease patient harboring a homozygous missense mutation (G364A) in the nuclear NDUFS7 gene (4). Expression of this specific mutation in Yarrowia lipolytica confirmed the deleterious effect on complex I enzyme activity (15). The human NDUFS7 protein is one of the most conserved subunits of complex I and plays a central role in the interaction with the electron acceptor ubiquinone and in the proton-translocating mechanism (16, 17). Here we show that altered cytosolic Ca2+ handling in complex I-deficient cells with a mutation in the NDUFS7 gene is associated with reduced mitochondrial Ca2+ accumulation and consequent ATP synthesis. An acute rescue of the defects was achieved following treatment with CGP37157 a benzothiazepine specifically inhibiting mitochondrial Na+-Ca2+ exchange.
ChemicalsCulture material was obtained from Invitrogen, and fluorescent dyes were from Molecular Probes Inc. (Leiden, The Netherlands). CGP37157was purchased from Tocris Cookson Ltd. (Avonmouth, Bristol, UK), and all other reagents were from Sigma. Patient FibroblastsFibroblasts were derived from skin biopsies of four healthy subjects and eight patients in the age range of 05 years in whom an isolated enzymatic complex I deficiency had been confirmed in both muscle tissue and cultured fibroblasts. The patient cells carried mutations the NDUFS1,2 NDUFS2 (1), NDUFS4 (2, 3), NDUFS7 (4), or NDUFV1 (6) gene. Skin fibroblasts were cultured in M199 medium containing 5 mg/liter Tween 20 and supplemented with 10% fetal calf serum, 100 IU/ml penicillin, and 100 IU/ml streptomycin.
Fluorescence Imaging of Cytosolic Ca2+Fibroblasts were seeded on 22-mm glass coverslips, grown to subconfluence for 24 h, and loaded with Fura-2 in the presence of 3 µM Fura-2/AM for 25 min at 37 °C. After removal of excess dye, the coverslip was mounted in a thermostatic (37 °C) perfusion chamber placed on the stage of an inverted microscope (Nikon Diaphot). Dynamic video imaging was carried out using the MagiCal hardware and Tardis software as described previously (18). Cells were perfused with Hepes-Tris medium (132 mM NaCl, 4.2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, 5.5 mM D-glucose, and 10 mM Hepes, pH 7.4), supplemented with an amino acid mixture according to Eagle, and challenged with 1 µM bradykinin to increase the cytosolic free Ca2+ concentration ([Ca2+]C). The Fura-2 fluorescence emission ratio at 492 nm was monitored as a measure of [Ca2+]C after alternating excitation at 340 and 380 nm. The kinetics with which the fluorescence emission ratio decreased was fitted to a monoexponential equation, R(t) = R(t = 0)·e Luminescence Monitoring of Cytosolic ATP, Endoplasmic Reticular Ca2+, and Mitochondrial Ca2+ and ATPBecause primary human skin fibroblasts are refractory to most conventional transfection protocols, we used an adenoviral system to express mitochondrially targeted aequorin (AdCMVmAq), mitochondrially targeted luciferase (AdCMVmLuc), and cytosolic luciferase (AdCMVcLuc) (19, 20), whereas a baculoviral system was used to express endoplasmic reticulum-targeted aequorin (BvCMVeAq). The latter system, which is normally used for protein production in Spodoptera frugiperda 9 insect cells, was made suitable for protein expression in mammalian cells by first removing the herpes simplex virus thymidine kinase polyadenylation signal from the pFastBacTMDual vector (Invitrogen) using AccI and XhoI, by next, after blunting and ligation, removing both the p10 and polyhedron promoter with SmaI and XbaI, and by replacing it with the coding region of a cytomegalovirus (CMV) promoter digested from the pcDNA1 vector (Invitrogen) using NruI and XbaI. Finally, the cDNA of ER-targeted aequorin was digested from the erAEQmut/pcDNA1 vector described by Montero et al. (21) with KpnI and NsiI and ligated behind the CMV promoter in the KpnI and PstI restriction sites of the modified baculovirus vector. Approximately 25,000 cells were spotted on a 13-mm coverslip, and after 24 h cells were infected with the appropriate virus and cultured for another 48 h. In case of baculoviral infection, the culture medium contained 1.75 mM sodium butyrate for proper expression of the photoprotein. Luciferase luminescence was monitored continuously using a photomultiplier tube (Thorn EMI Electron tubes, Ruislip, Middlesex, UK) (19). Cells were perfused (2 ml·min1) with modified Krebs-Ringer bicarbonate medium (KRB; 140 mM NaCl, 3.5 mM KCl, 0.5 mM NaH2PO4, 0.5 mM MgSO4, 10 mM Hepes, 2 mM NaHCO3, 1 mM CaCl2, 5.5 mM D-glucose, pH 7.4) containing 5 µM beetle luciferin (Promega, Madison, WI) at 37 °C. Emitted light was collected with a photoncounting board using the supplier's software (Thorn EMI) (22). Light output was recorded at 1-s intervals after which the traces were smoothed off-line by using a 5-point moving average (Origin Pro 6.1, OriginLab Corporation, Northampton, MA). Typically, light output from a coverslip of virally infected fibroblasts was 1,00025,000 counts·s1 versus a background of 10 counts·s1. The same system was used to monitor aequorin luminescence. Mitochondrial aequorin was reconstituted with 5 µM coelenterazine (Molecular Probes) in KRB for 1 h at 37 °C. Light output was recorded at 1-s intervals, and at the end of each experiment the signal was calibrated by lysing the cells with 100 µM digitonin and 10 mM CaCl2 to determine the total photoprotein content. Aequorin photon emission was converted off-line into [Ca2+] values using a computer algorithm based on the Ca2+ response curve of wild-type aequorin (23). For measurements of endoplasmic reticulum aequorin, we first reduced the Ca2+ content of this organelle by incubating the cells with a reversible inhibitor of the ER Ca2+-ATPase, BHQ (10 µM), in Ca2+-free KRB (no CaCl2 added and 0.5 mM EGTA present) for 10 min. After washing, endoplasmic reticulum aequorin was reconstituted with 5 µM coelenterazine n (Molecular Probes) in Ca2+-free KRB containing 10 µM BHQ for 1.5 h at room temperature. Next, the glass coverslip was placed in the luminometer, and cells were initially perfused with Ca2+-free KRB for 5 min at 37 °C to remove BHQ. To determine the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)-mediated Ca2+ uptake activity, cells were perfused with a Ca2+-free intracellular medium (10 mM Hepes, 120 mM KCl, 5 mM NaCl, 2.5 mM MgCl2, 2 mM EGTA, 2.5 mM ATP, pH 7.05) containing 20 µg/ml saponin for 2 min at 37 °C to selectively permeabilize the plasma membrane (24). SERCA-mediated Ca2+ uptake into the ER was started by perfusing with intracellular medium containing 0.55 mM CaCl2 (free Ca2+ concentration of 0.1 µM). The luminescence data were calibrated as described previously by Alvarez and Montero (25).
Fluorescence Imaging of Mitochondrial Membrane Potential (
Determination of ATP Levels in Cell HomogenatesPellets containing Data AnalysisNumerical values were visualized using Origin Pro 6.1 (OriginLab Corporation), and values from multiple experiments were expressed as average ± S.E. Statistical significances were assessed by Student's t test.
Impaired Cytosolic Ca2+ Handling in Complex I-deficient Human FibroblastsThe present study was aimed at establishing the consequences of complex I deficiency on intracellular Ca2+ homeostasis in the context of the genetic background of the patient. Skin fibroblasts from four control individuals and eight patients with isolated enzymatic complex I deficiency caused by nuclear DNA mutations were loaded with the fluorescent Ca2+ indicator Fura-2 for monitoring of the bradykinin-induced changes in [Ca2+]C in individual cells using digital imaging microscopy. An increase in [Ca2+]C is reported by an increase in the ratio of the fluorescence emission intensities at excitation wavelengths of 340 and 380 nm.
Control fibroblasts displayed resting emission ratios of 0.48 ± 0.02 (n = 24), 0.48 ± 0.02 (n = 31), 0.54 ± 0.03 (n = 30), and 0.52 ± 0.02 (n = 29) that were not significantly different between the four individuals. Immediately after stimulation with bradykinin (1 µM) the cells displayed a sharp rise in [Ca2+]C (see also Fig. 3D). Maximum increases in the emission ratio were 4.47 ± 0.15 (n = 24), 4.71 ± 0.19 (n = 31), 4.89 ± 0.19 (n = 30), and 4.74 ± 0.15 (n = 29) x base line and did not statistically differ. The amplitude of the [Ca2+]C rise was independent of extracellular Ca2+, demonstrating that Ca2+ ions entered the cytosolic compartment from the endoplasmic reticular Ca2+ store. After having reached its maximum, [Ca2+]C more gradually declined again to prestimulatory levels. The decrease in fluorescence emission ratio was fitted monoexponentially, and half-times (t
Patient and control fibroblasts did not differ with respect to the resting [Ca2+]C (see also Fig. 3D). However, the bradykinin-induced peak increase in [Ca2+]C was significantly decreased in four patients, whereas the rate of cytosolic Ca2+ removal was significantly, but to a variable degree, slowed down in all patients as indicated by an increase in half-time (Fig. 1). Fibroblasts with a mutation in either the NDUFS4 (Fig. 1, C316T; n = 23 and n = 18 for patients a and b, respectively) or NDUFS7 (G364A; n = 21) gene displayed both a marked reduction in peak [Ca2+]C ( 20%; p < 0.001) and a large increase in t (1.6-fold; p < 0.001). The NDUFS1 mutation (C1668T/G1854A; n = 16) showed a moderate reduction in peak height ( 10%; p < 0.05) and a moderate increase in t (1.3-fold; p < 0.001), whereas the two NDUFS2 mutations, C686A (n = 20) and T1237C (n = 22), displayed only a moderate increase in t (1.4- and 1.3-fold, respectively; p < 0.001). Finally, the NDUFS4 (471 AAGTC insertion; n = 31); and NDUFV1 (C175T/C1268T; n = 20) mutations displayed a small increase in t (1.1-fold; p < 0.05) and no change in peak height.
A significant (p < 0.01) correlation was observed between peak height and Ca2+ removal rate, indicating a common cause for their reduction in human complex I deficiency (Fig. 1). Because of the marked effects observed with fibroblasts carrying a G364A mutation in the NDUFS7 gene, we used these cells in a series of experiments conducted to gain insight into the mechanism(s) underlying the changes in cytosolic Ca2+ handling in human complex I deficiency.
Reduced Filling State of the Endoplasmic Reticular Ca2+ Store in Unstimulated Complex I-deficient FibroblastsThe bradykinin-induced [Ca2+]C transients in fibroblasts are likely to be shaped mainly by the relative rates of (i) Ca2+ release from the ER into the cytoplasm, (ii) Ca2+ uptake and subsequent release by mitochondria, and (iii) cytosolic Ca2+ removal via SERCAs and plasma membrane Ca2+-ATPases. To investigate whether the ER Ca2+ content is altered in resting patient fibroblasts with a G364A mutation in the NDUFS7 gene, cells were loaded with Fura-2 and subsequently treated with the Ca2+ ionophore ionomycin. Measurements were performed in the absence of extracellular Ca2+ to prevent capacitative Ca2+ entry in response to Ca2+ store depletion. Ionomycin (1 µM) transiently increased [Ca2+]C in both control and patient fibroblasts (Fig. 2A). The area under the peak was
To determine possible differences in SERCA number and/or transport properties, fibroblasts expressing ER-targeted aequorin were first permeabilized with saponin in a Ca2+-free intracellular medium containing 2.5 mM ATP. Next, SERCA-mediated Ca2+ uptake was started by increasing the free [Ca2+] of the perfusion medium to 0.1 µM, which equals the cytosolic free [Ca2+] in unstimulated cells. Both control and patient fibroblasts showed a rapid increase in [Ca2+]ER (Fig. 2B). The maximal rate of [Ca2+]ER increase was not different between control (13 ± 1 µM·s1, n = 6) and patient (12 ± 1 µM·s1, n = 6) fibroblasts. Similarly, the peak [Ca2+]ER, which was reached after 60 s, was not different between control (442 ± 22 µM, n = 6) and patient fibroblasts (449 ± 17 µM, n = 6). These findings demonstrate that under conditions in which ATP is not rate-limiting, both the SERCA activity and the physical size of the ER Ca2+ store are the same for control and patient fibroblasts. To address the possibility that Ca2+ leakage from the ER is increased in patient fibroblasts, cells were loaded with Fura-2 and subsequently treated with the SERCA inhibitor BHQ in the absence of extracellular Ca2+. Both in control and patient fibroblasts, BHQ (10 µM) evoked an immediate increase in [Ca2+]C (Fig. 2C). The maximal rate of [Ca2+]C increase was not significantly different between control (0.016 ± 0.001 arbitrary unit·s1, n = 13) and patient (0.016 ± 0.001 arbitrary unit·s1, n = 16) fibroblasts. In contrast, the peak [Ca2+]C was significantly (p < 0.001) lower in patient (1.42 ± 0.01 x base line) as compared with control fibroblasts (1.58 ± 0.01 x base line). The latter finding is compatible with a decreased ER Ca2+ content in resting complex I-deficient fibroblasts. Finally, measurement of the total ATP content in whole cell lysates using a luciferase-based assay revealed a small (5%) but statistically insignificant decrease in patient fibroblasts (p = 0.08; n = 4 for both control and patient fibroblasts).
Decreased Mitochondrial Ca2+ Accumulation in Bradykinin-stimulated Complex I-deficient FibroblastsIt has been shown previously that agonist-induced increases in [Ca2+]C lead to parallel increases in the total (26) and free (27, 28) concentration of Ca2+ in mitochondria ([Ca2+]M). To investigate whether mitochondrial Ca2+ accumulation is altered in complex I deficiency, patient fibroblasts were infected with adenoviruses expressing mitochondrially targeted aequorin. The cells ( Pretreatment of the cells with the protonophore FCCP (1 µM, 2 min) completely abolished the bradykinin-induced increase in [Ca2+]M. In contrast, the drug inhibited the bradykinin-induced peak increase in [Ca2+]C only by 25% (see also Fig. 4A). This demonstrates that aequorin was exclusively present in the mitochondrial compartment.
Reduced Mitochondrial Membrane Potential in Complex I-deficient FibroblastsStudies using suspensions of isolated mitochondria have suggested that mitochondrial Ca2+ uptake is mediated by an electrogenic "low affinity" uniporter (K0.5 = 510 µM) driven by the large electrical gradient ( ![]() M 160 mV) created by the respiratory chain (29, 30). However, by patch-clamping vesicles of the inner mitochondrial membrane, Clapham and co-workers (31) were able to demonstrate recently that the mitochondrial Ca2+ uptake system is, in fact, a highly selective Ca2+ channel with a half-activation constant of 19 mM. It has been demonstrated that a decrease in respiratory activity results not only in a reduction in ![]() M (10) but also in a decrease in agonist-stimulated mitochondrial Ca2+ uptake (12). To assess whether the ![]() M is decreased in complex I-deficient cells, patient fibroblasts were loaded with the fluorescent dye JC-1, which accumulates within the mitochondrial matrix as a function of ![]() M (32). At high concentrations this dye forms J-aggregates with a red fluorescence emission signal. Counting of the number of red objects/cell revealed a significant (p < 0.01) difference between patient (89 ± 3, n = 42) and control (104 ± 3, n = 39) fibroblasts. Pretreatment with 1 µM FCCP for 2 min reduced the amount of J-aggregates to 4 ± 1 (n = 11), demonstrating their dependence on ![]() M.
Reduced Mitochondrial ATP Production in Bradykinin-stimulated Complex I-deficient FibroblastsAgonist-induced increases in [Ca2+]M have been shown previously to cause activation of intramitochondrial dehydrogenases (33, 34) and consequent increases in NAD(P)H and FADH2 (35, 36). This in turn stimulates respiratory chain activity and mitochondrial ATP synthesis (12, 37). To investigate whether mitochondrial ATP production is altered in complex I deficiency, patient fibroblasts were infected with adenoviruses expressing mitochondrially targeted or cytosolic firefly luciferase. Bradykinin evoked a gradual increase in both [ATP]M (Fig. 3B) and [ATP]C (Fig. 3C) that lasted
Fig. 3D compares the bradykinin-induced changes in [Ca2+]C between Fura-2-loaded control and patient fibroblasts. In both cases, the peak increase in [Ca2+]C was observed
Restoration of Impaired Ca2+ and ATP Homeostasis by CGP37157in Human Complex I-deficient FibroblastsIt has been demonstrated that cells of a cybrid cell line of Next, we addressed the question of whether restoration of mitochondrial Ca2+ uptake resulted in enhanced activation of the OXPHOS system. In patient fibroblasts, CGP37157increased both the rising speed and the peak value of the bradykinin-induced [ATP]M increase to control values (rising speed of 0.31 ± 0.04%·s1 and peak value of 107.8 ± 0.6%, n = 5) (Fig. 3F). The drug did not affect these parameters in control cells (rising speed of 0.37 ± 0.03%·s1 and peak value of 109.4 ± 0.6%, n = 4). The restoration of agonist-induced mitochondrial ATP production was accompanied by an increase in rising speed and peak value of the [ATP]C increase (rising speed of 0.17 ± 0.01%·s1 and peak value of 104.8 ± 0.3%, n = 4) (Fig. 3G). The drug did not significantly alter these parameters in control cells (rising speed of 0.18 ± 0.01%·s1 and peak value of 105.3 ± 0.4%, n = 4).
Finally, we investigated the effect of this drug on the bradykinin-induced [Ca2+]C rise in Fura-2-loaded patient fibroblasts. CGP37157completely restored both the bradykinin-induced peak increase in [Ca2+]C and the t
CGP37157-induced Restoration of Cytosolic Ca2+ Handling in Complex I-deficient Fibroblasts Depends on the Mitochondrial Membrane PotentialTo investigate the possible involvement of extracellular Ca2+ influx in the mechanism of action of CGP37157 Fura-2-loaded cells were stimulated in the absence of external Ca2+ (no Ca2+ added and 0.5 mM EGTA present). Also under these conditions patient fibroblasts displayed a decrease in bradykinin-induced peak [Ca2+]C (Fig. 4A) and an increase in t
The importance of Regarding the effect of FCCP on [Ca2+]ER, only a small reduction was observed at 2 min after addition of the drug (Fig. 4C). In control cells this reduction was 11% (n = 22), whereas in patient cells this reduction was 5% (n = 28).
With respect to the bradykinin-induced [Ca2+]C transient, pretreatment of control cells with 1 µM FCCP for 2 min significantly (p < 0.001) decreased the peak increase in [Ca2+]C by
Human complex I (NADH:ubiquinone oxidoreductase) deficiency leads to a wide variety of clinical disease presentations, of which the underlying cell biological causes are poorly understood (7). Here we show that mitochondrial membrane potential and, as a consequence, agonist-induced mitochondrial Ca2+ uptake and ensuing stimulation of mitochondrial ATP production are impaired in skin fibroblasts from a patient with Leigh disease carrying a homozygous missense mutation (G364A) in the nuclear NDUFS7 gene (4). We also show that the impairments in mitochondrial function are associated with a reduced filling state of the ER Ca2+ store, a decreased agonist-induced peak [Ca2+]C increase, and a reduced rate of cytosolic Ca2+ removal following agonist stimulation. Given the unaltered capacity for ER Ca2+ uptake it seems unlikely that the reduced filling state of the ER Ca2+ store is because of changes in its physical size and/or the number and transport properties of the SERCA Ca2+ pumps; rather, the reduced filling state is secondary to changes in cellular ATP synthesis. Unexpectedly, we show that cytosolic ATP levels are lower in patient versus control cells even in the presence of high concentrations of a mitochondrial uncoupler, suggestive of extramitochondrial changes in ATP synthetic capacity. Although an exploration of the nature of these changes is beyond the scope of the present work, it is conceivable that decreases in glycolytic enzyme activity, enhanced activity of ATP-consuming mechanisms, or both are involved and represent cellular consequences of the decrease in mitochondrial oxidative capacity. Importantly, all aberrations, except for the decreased filling state of the ER, were completely restored upon acute inhibition of mitochondrial Na+-Ca2+ exchange by the benzothiazepine CGP37157 presumably acting to restore agonist-induced mitochondrial ATP synthesis and thus overcoming small differences in extramitochondrial ATP synthesis or consumption. Growing evidence suggests that under certain circumstances agonist-induced Ca2+ signals can turn from a survival signal into a death signal (39). For instance, agonist-induced mitochondrial Ca2+ uptake may induce apoptosis by activation of the permeability transition pore in response to a variety of pathological conditions (40). The cellular and molecular mechanisms underlying this switch are just beginning to be understood. Thus, enhanced formation of superoxide and hydrogen peroxide has been demonstrated to promote the Ca2+-dependent opening of the permeability transition pore (41). Because the production of reactive oxygen species is increased in complex I-deficient cells (9), it is of importance to have detailed information about cytosolic and mitochondrial Ca2+ handling in cells with a malfunctioning complex I, especially under conditions of increased cytosolic Ca2+ mobilization as they occur during hormonal and electrical stimulation.
Decreased Agonist-induced Mitochondrial Ca2+ Uptake and Ensuing ATP Production Are Restored by CGP37157in Complex I-deficient FibroblastsUnder non-pathological conditions, agonist-induced mitochondrial Ca2+ uptake leads to an increase in mitochondrial ATP production (12, 37) through the activation of mitochondrial dehydrogenases (35). Here we show that bradykinin-induced mitochondrial Ca2+ uptake and ATP production are decreased in fibroblasts from a patient with isolated complex I deficiency. Importantly, CGP37157restored the bradykinin-induced increase in both [Ca2+]M and [ATP]M, indicating that the reduction in mitochondrial ATP production observed in agonist-stimulated patient fibroblasts is because of a decrease in mitochondrial Ca2+ uptake. The reduction of the bradykinin-induced peak increase in [Ca2+]M was only
Reduced Agonist-induced Increase in [Ca2+]C Is Restored by CGP37157in Complex I DeficiencyStimulations performed in the absence of external Ca2+ revealed that bradykinin increases [Ca2+]C and, as a consequence, [Ca2+]M by promoting the release of Ca2+ from the ER in human skin fibroblasts. Here we show that the filling state of the ER is significantly reduced in patient fibroblasts. The data presented show that this reduction is not because of a decrease in physical size of the store. Neither is this reduction because of a decrease in SERCA number and/or transport properties or an increase in Ca2+ leakage. Our finding that the cytosolic ATP level and, as a consequence, the rate of cytosolic Ca2+ removal are lower in FCCP-treated patient cells as compared with FCCP-treated control cells indicates that in patient cells glycolytic activity is decreased and/or that ATP consumption is increased. In untreated patient cells, these secondary effects of complex I deficiency may contribute to a decrease in SERCA-mediated filling of the ER Ca2+ store. Measurements performed with JC-1, a fluorescent dye that accumulates within the mitochondrial matrix as a function of It can be hypothesized that bradykinin releases less Ca2+ from the ER in patient fibroblasts and therefore evokes a reduced increase in [Ca2+]C and, as a consequence, [Ca2+]M in these cells. However, the present study shows that in untreated patient fibroblasts the ER Ca2+ content and bradykinin-induced peak increase in [Ca2+]C were decreased by 21 and 13%, respectively, whereas in FCCP-treated control fibroblasts these values were 11 and 23%, respectively. Importantly, this finding indicates that the decrease in [Ca2+]C observed following acute FCCP treatment cannot be because of a decrease in ER Ca2+ content alone. It has been demonstrated that the activity of the inositol 1,4,5-trisphosphate (InsP3) receptors that mediate the agonist-induced release of Ca2+ from the ER is regulated by ambient [ATP]C and [Ca2+]C (42). Decreases in [ATP]C have been shown to activate InsP3-induced Ca2+ release. Here we show, however, that acute FCCP treatment, despite causing a dramatic decrease in [ATP]C, decreased rather than increased the bradykinin-induced increase in [Ca2+]C. Other studies have shown that the activity of InsP3 receptors is inhibited at high ambient [Ca2+]C (28, 4345). This opens the possibility that FCCP, by abolishing mitochondrial Ca2+ uptake, causes a buildup of the cytosolic Ca2+ concentration in the mouth of the InsP3-operated Ca2+ channels to levels that inhibit the Ca2+ release process, thereby reducing the total amount of Ca2+ that is released from the ER into the cytosol (46). It should be noted that this buildup of the cytosolic Ca2+ concentration during agonist stimulation is likely to be enhanced by the dramatic reduction in [ATP]C and consequent decrease in SERCA-mediated Ca2+ uptake into the ER.
FCCP abolishes both the
The finding that inhibition of mitochondrial Na+-Ca2+ exchange restores the peak increase in [Ca2+]C provides us with intriguing information about the interplay between neighboring ER Ca2+ channels and mitochondrial Ca2+ uptake and extrusion sites (47). According to the above hypothesis, the restorative action of CGP37157in patient fibroblasts can be explained in that the drug by inhibiting mitochondrial Ca2+ release enhances the bradykinin-induced increase in [Ca2+]M and consequent increases in dehydrogenase activity,
Decreased Cytosolic Ca2+ Removal following Agonist Stimulation and Its Restoration by CGP37157in Complex I DeficiencyA significant correlation was observed between the rate of cytosolic Ca2+ removal and the peak increase in [Ca2+]C in bradykinin-stimulated patient fibroblasts, indicating a common cause for their reduction in human complex I deficiency. Similarly to four of the eight patient cell lines studied here, the agonist-induced peak increase in [Ca2+]C was not altered in a cybrid cell line of As a consequence of decreased agonist-induced mitochondrial ATP production, fueling of energy-requiring processes, set in motion by the increase in [Ca2+]C, becomes jeopardized. One of these processes is the energy-dependent extrusion of Ca2+ from the cytosolic compartment. Indeed, the present study shows that this process is significantly slowed down in bradykinin-stimulated patient fibroblasts. We demonstrated recently that the rate of sarcoplasmic Ca2+ removal following electrical stimulation was significantly reduced in myotubes cultured from the quadriceps muscle of patients with an adult onset of exercise intolerance and exercise-induced myalgia and stiffness exhibiting a biochemically defined decrease in mitochondrial ATP production capacity (48). It is tempting to speculate that in the long term, the cumulative effect of such longer lasting [Ca2+]C rises is toxic to the cell. It has been demonstrated that sustained elevations of [Ca2+]C can switch on a number of mechanisms leading to necrotic as well as apoptotic cell death (4951). The present finding that CGP37157can normalize the rate of cytosolic Ca2+ removal by restoring mitochondrial ATP synthesis in complex I-deficient patient fibroblasts may open possibilities of future therapeutic treatment. In this context, it is of importance to realize that the drug did not affect the rate of cytosolic Ca2+ removal in fibroblasts from a healthy subject. ConclusionsIntriguingly, although the OXPHOS system contains a defective complex I, it was possible to enhance mitochondrial ATP synthesis by acute treatment with CGP37157 It is of note that enzymatic deficiencies in different parts of the energy-producing cascade, like the pyruvate dehydrogenase complex, complex I, and complex IV of the OXPHOS system, may all lead to Leigh disease. It is therefore tempting to assume that Leigh disease, although genetically different, has a similar underlying pathophysiological basis of which Ca2+ may be the common denominator.
* This work was supported by 6th Framework Programma Integrated Project Grant LSHM-CT-2004-503116 from the European Community and a Van Walree Fund grant from the Royal Netherlands Academy of Arts and Sciences. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶¶ To whom correspondence should be addressed: Nijmegen Center for Mitochondrial Disorders, Dept. of Pediatrics, University Medical Center Nijmegen, P. O. Box 9101, NL-6500 HB Nijmegen, The Netherlands. Tel.: 31-243614430; Fax: 31-243616428; E-mail: j.smeitink{at}cukz.umcn.nl.
1 The abbreviations used are: OXPHOS, oxidative phosphorylation; NDUFS7, NADH dehydrogenase ubiquinone flavoprotein S7 subunit; BHQ, 2,5-di-tert-butyl-benzohydroquinone; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; MELAS, myopathy, encephalopathy, lactic acidosis, stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fibers; [Ca2+]C, [Ca2+]ER, and [Ca2+], cytosolic, endoplasmic reticular, and mitochondrial free Ca2+ concentrations; [ATP]C and [ATP]M, cytosolic and mitochondrial ATP concentrations; ER, endoplasmic reticulum; InsP3, inositol 1,4,5-trisphosphate; CMV, cytomegalovirus; KRB, Krebs-Ringer bicarbonate medium; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase.
2 J. A. M. Smeitink and L. P. van den Heuvel, unpublished data.
We thank Dr. Gabriela Da SilvaXavier (University of Bristol), Dr. Takashi Tsuboi (University of Bristol), and Martijn Wilmer (Department of Pediatrics, University Medical Center Nijmegen) for technical assistance.
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