Published online before print April 27, 2009, doi: 10.1161/CIRCULATIONAHA.108.835389
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(Circulation. 2009;119:2435-2443.)
© 2009 American Heart Association, Inc.
Arrhythmia/Electrophysiology |
Regulation of the Human Cardiac Mitochondrial Ca2+ Uptake by 2 Different Voltage-Gated Ca2+ Channels
From the Department of Internal Medicine III (G.M., I.F.K., J.E.-B., D.R., U.C.H.), Center for Molecular Medicine (S.H., U.C.H.), Institute of Pharmacology (S.H.), and Department of Cardiothoracic Surgery (T.W.), University of Cologne, Cologne, and Department of Cardiac Surgery, University of Heidelberg, Heidelberg (A.R.), Germany.
Correspondence to Uta C. Hoppe, MD, Department of Internal Medicine III, University of Cologne, Kerpener-Str 62, 50937 Cologne, Germany. E-mail uta.hoppe{at}uni-koeln.de
Received July 13, 2008; accepted March 9, 2009.
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Abstract |
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Background— Impairment of intracellular Ca2+ homeostasis and mitochondrial function has been implicated in the development of cardiomyopathy. Mitochondrial Ca2+ uptake is thought to be mediated by the Ca2+ uniporter (MCU) and a thus far speculative non-MCU pathway. However, the identity and properties of these pathways are a matter of intense debate, and possible functional alterations in diseased states have remained elusive.
Methods and Results— By patch clamping the inner membrane of mitochondria from nonfailing and failing human hearts, we have identified 2 previously unknown Ca2+-selective channels, referred to as mCa1 and mCa2. Both channels are voltage dependent but differ significantly in gating parameters. Compared with mCa2 channels, mCa1 channels exhibit a higher single-channel amplitude, shorter openings, a lower open probability, and 3 to 5 subconductance states. Similar to the MCU, mCa1 is inhibited by 200 nmol/L ruthenium 360, whereas mCa2 is insensitive to 200 nmol/L ruthenium 360 and reduced only by very high concentrations (10 µmol/L). Both mitochondrial Ca2+ channels are unaffected by blockers of other possibly Ca2+-conducting mitochondrial pores but were activated by spermine (1 mmol/L). Notably, activity of mCa1 and mCa2 channels is decreased in failing compared with nonfailing heart conditions, making them less effective for Ca2+ uptake and likely Ca2+-induced metabolism.
Conclusions— Thus, we conclude that the human mitochondrial Ca2+ uptake is mediated by these 2 distinct Ca2+ channels, which are functionally impaired in heart failure. Current properties reveal that the mCa1 channel underlies the human MCU and that the mCa2 channel is responsible for the ruthenium red–insensitive/low-sensitivity non-MCU–type mitochondrial Ca2+ uptake.
Key Words: calcium • electrophysiology • heart failure • mitochondria • ion channels
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Introduction |
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Mitochondrial Ca2+ transport was discovered in the early 1960s.1 Mitochondria have been shown to dominate in the clearance of cytosolic Ca2+ in different cells and are able to accumulate a large amount of Ca2+.2 The mitochondrial Ca2+ uptake from the cytosol controls the rate of energy production,3,4 modulates the spatial and temporal profile of intracellular Ca2+ signaling,5–9 regulates mitochondrial motility and morphology,10 and may trigger cell death.11 The Ca2+ transport into mitochondria is mediated by the Ca2+ uniporter (MCU), which is blocked by nanomolar concentrations of ruthenium red (RuR) or its analog, ruthenium 360 (Ru360), located in the inner mitochondrial membrane.2,12–15
Clinical Perspective on p 2443
Mitochondrial Ca2+ uptake has been evaluated indirectly for several years, ie, by Ca2+ sensors or measurement of Ca2+ gradients,6,12,16 allowing only limited experimental accuracy. Kirichok et al15 were able to directly record a highly selective mitochondrial Ca2+ current, which is driven by the negative mitochondrial potential, activated by extramitochondrial Ca2+ (half-activation constant, 
20 mmol/L; Ymax, 105 mmol/L) and inhibited by 200 nmol/L RuR or Ru360, thus exhibiting characteristics of the MCU. Because these recordings were performed in mitoplasts (2- to 5-µm vesicles of inner mitochondrial membrane) from COS-7 cells, results may not readily be transferred to the human setting. Besides the classic MCU, the presence of a second RuR-insensitive mitochondrial Ca2+ uptake pathway, which is inhibited only by very high RuR concentrations (>1 µmol/L), has been hypothesized.2,12,17 Moreover, it is of particular interest whether mitochondrial Ca2+ channel function is impaired in diseased states associated with profound alterations of intracellular Ca2+ homeostasis and metabolism such as heart failure.18
Therefore, our goal was to directly record and identify channels underlying Ca2+ influx of human mitochondria. Second, we intended to evaluate the impact of end-stage heart failure on human mitochondrial Ca2+ uptake. Our results demonstrate the existence of 2 different selective Ca2+ channels of the human cardiac inner mitochondrial membrane. Furthermore, both mitochondrial Ca2+ channels are dysfunctional under heart failing conditions.
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Methods |
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Patients
Human cardiac mitoplasts were isolated from 15 hearts of patients with end-stage heart failure caused by dilated cardiomyopathy or ischemic cardiomyopathy who were undergoing transplantation. Patient characteristics are given in Table I of the online Data Supplement. Results were compared with mitoplasts prepared from 3 human hearts without heart failure that could not be transplanted for technical reasons (possible systemic infectious disease [n=1], blood group incompatibility [n=2]). Informed consent was obtained before organ transplantation or donation. The study was approved by the local ethics committee.
Preparation of Myocytes and Mitoplasts
Single human ventricular myocytes were isolated from nonfailing and failing hearts by enzymatic digestion, as previously described.19 Freshly isolated myocytes were used within 1 to 4 hours. Isolated intact mitoplasts were prepared from isolated myocytes by differential centrifugation, as previously reported.15,20 For patch-clamp studies, myocytes were labeled with Mitotracker Green 1 µmol/L (Molecular Probes, Inc, Carlsbad, Calif; Figure 1A and Table) to facilitate identification of intact mitoplasts after further subcellular purification.
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Single-Organelle/Cell Reverse-Transcription Polymerase Chain Reaction Analysis
cDNA synthesis and polymerase chain reaction (PCR) were performed with the OneStep reverse-transcription PCR kit (Qiagen, Hilden, Germany) with some variation from the manufacturer’s directions: 2 isolated mitoplasts or cardiomyocytes were pooled in 20 µL of 1x reaction buffer. Master mix was prepared as specified in the manual and added to a total reaction volume of 55 µL in 0.5-mL thin-walled PCR tubes. The mixture was incubated at 50°C for 30 minutes. After cDNA synthesis, the reaction was distributed into 0.2-mL PCR tubes preloaded with 5 pmol (each) sense and antisense primer specific for mitochondrial cytochrome C oxidase I (sense, 5'-CTC CCA CCC TGG AGC CTC CGT AGA C-3'; antisense, 5'-GGG AGA TTA TTC CGA AGC CTG GTA G-3'; amplifying 358 bp) and GAPDH (sense, 5'-GGT CGG TGT GAA CGG ATT TG-3'; antisense, 5'-GTG AGC CCC AGC CTT CTC CAT-3'; amplifying 318 bp). Amplification was performed in a Thermocycler gradient (Eppendorf, Hamburg, Germany), starting at 95°C for 15 minutes to deactivate the reverse transcriptase and to activate the Taq polymerase. The program consisted of 40 cycles (denaturation, 95°C for 30 seconds; annealing, 68°C gradient for 45 seconds; elongation, 72°C for 1 minute) with a final elongation step at 72°C for 10 minutes. Samples were analyzed on 1.5% agarose gels, stained with ethidium bromide, and documented on a BioDocAnalyze 2.0 (Biometra, Göttingen, Germany).
Single-Channel Recordings
All experiments were performed in the mitoplast-attached configuration of the patch-clamp technique (test pulse, 150-ms duration at 1.67 Hz; sampling frequency [fs], 10 kHz; cutoff frequency [fc], 2 kHz), as previously described.20 Only experiments with a high seal resistance between 40 and 80 G
were included. The bath solution contained 160 mmol/L KCl, 10 mmol/L HEPES, 1 mmol/L EDTA, and 1 mmol/L EGTA (pH 7.2 with KOH). Pipettes were filled with a solution composed of 105 mmol/L CaCl2 (if not otherwise indicated), 10 mmol/L HEPES [pH 7.2 with Ca(OH)2]. Specific drugs were added to solutions to block the mitochondrial permeability transition pore (cyclosporin A, Sigma Aldrich, St Louis, Mo; 10 µmol/L), the mitochondrial ryanodine receptor (dantrolene, Sigma Aldrich; 10 µmol/L), the IP3R (xestospongin C, Sigma Aldrich; 10 µmol/L), the mitochondrial Na+-Ca2+ exchanger (CGP-37157, Calbiochem, San Diego, Calif; 10 µmol/L), and the mCa1/2 (Ru360, Calbiochem; 200 nmol/L or 10 µmol/L) as indicated. Spermine (Sigma Aldrich; 1 mmol/L) was added to activate mCa1/2, and S(-)-BayK 8644 (Sigma Aldrich; 10 µmol/L) was added to rule out that mitochondrial Ca2+ channels are 1,4-dihydropyridine sensitive. Currents were recorded and digitized with an Axopatch 200B amplifier and Digidata 1200 interface (Axon Instruments, Foster City, Calif).
Single-Channel Data Analysis
Single-channel analysis was done with custom software (CDE-REVL-LEVL/X program, version 1.3), as previously reported21 and further extended for this work (supplemental Methods). Linear leak and capacity currents were digitally subtracted using the average currents of nonactive sweeps. For detailed gating analysis, idealized currents were analyzed in 150-ms steps. The open probability (defined as the occupancy of the open state during active sweeps), the availability (fraction of sweeps containing at least 1 channel opening), and Ipeak (the peak ensemble average current, either fitted by an adapted Hodgkin-Huxley model if possible [continuously differentiable] or computed by the local minimum method, as given previously21) were calculated only from single-channel patches. To determine single-channel amplitudes most accurately, amplitudes were obtained not only by direct measurements of visually fully resolved openings and as the maximum of gaussian fits on amplitude histograms but also by the use of a peak integral differential detector (supplemental Methods). Despite this evaluation, we may not entirely exclude that the full conductance of channels with spike-like gating kinetics might have been underestimated.
Sublevel Analysis of Single-Channel Data
PMAX (maximum of simultaneously open channels) is an estimator for n, the exact number of channels in a patch with k simultaneous detected openings (stacked current levels). PMAX is reliable only for patches where k
5, as included here for analysis (CDE-REVL-LEVL/X program, version 1.3). The PMAX probability, Prob (channel n=k), is then given by the following: Prob (n=k)=PMAX (k, Po, M)=1–(1–Pok)M, where k is the number of simultaneous openings or sublevels, n is the real number of channels or sublevels, Po is the single-channel open probability calculated over the entire recording time, and M is the total number of sweeps.22
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Patient Characteristics
To directly analyze the Ca2+ uptake in human mitochondria and to assess the impact of heart failure on mitochondrial Ca2+ channel function, we patch clamped single cardiac mitoplasts (Figure 1A and 1B) of 18 human hearts (supplemental Table I). To omit any contamination by mitochondria of nonmyocytes, we performed a 2-step isolation process, ie, isolation of human cardiomyocytes, which then were used for mitoplast generation.
Human Mitochondrial Ca2+ Uptake Is Regulated by 2 Different Mitochondrial Ca2+ Channels
Consistent with previous single Ca2+ channel recordings of mitoplasts from COS-7 cells,15 we also chose a [Ca2+]pipette of 105 mmol/L. In the mitoplast-attached configuration, voltage steps to –100 mV elicited 2 distinct types of voltage-dependent Ca2+ currents (Figure 1C and 1D) that we refer to as ImCa1 and ImCa2 (for mitochondrial Ca2+ current type 1 and 2). Occasionally, both Ca2+ currents could be recorded in same patches (Figure 1E), indicating that mCa1 and mCa2 channels are not separately present and thus characteristic for different mitochondrial subpopulations.23 These 2 Ca2+ currents differed in several single-channel parameters (the Table and Figure 1C and 1D). Compared with mCa1 channels, mCa2 channels had a smaller single-channel amplitude, longer openings (mean open times), and a higher open probability. Moreover, mCa1 and mCa2 channels exhibited a distinct bursting behavior (mCa2>mCa1; supplemental Table II). Activity of both mitochondrial Ca2+ channels increased at more negative voltages (higher open probability and availability, shorter mean first latency) (Figure 2C), making these channels effective for Ca2+ uptake into energized mitochondria. Single-current amplitudes of ImCa1 and ImCa2 increased at more negative potentials, with mCa1 channels exhibiting a significantly higher conductance compared with mCa2 channels (nonfailing: gmCa1 13.7±0.78 pS [n=3] versus gmCa2 7.67±0.88 pS [n=3]; P<0.05) (Figure 2A and the Table). To exclude the possibility that our biophysical analysis of mCa1 versus mCa2 channels might be biased by a selection artifact, we plotted two of the proposed distinctive biophysical features, ie, single-channel conductance and mean closed time (at Vm –100 mV), against each other (Figure 2B). No single case of overlap based on these 2 independent parameters further supports the distinct identity of the 2 Ca2+ currents.
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To show that the amplitudes of mCa1 taken as peaks are averages of multiple points (
1 sample point), we performed additional recordings at higher bandwidth (sampling frequency, 25 kHz; cutoff frequency, 5 kHz) (Figure 3A). However, a wider bandwidth of 5 kHz did not provide any improvement in single-channel detection in unfiltered data or its post–digital-filtered correlate (8-pole Bessel filter; fc=2 kHz) compared with 2-kHz analog-filtered data. Rather, a higher filter frequency of 5 kHz created a higher root mean square background noise, a reduced signal-to-noise ratio, a higher number of spurious and false events, and digital postfiltered sweeps, which were rarely distinguishable from their 2-kHz analog-filtered counterparts.
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Gating and Permeability of Both Mitochondrial Ca2+ Channels Are Suppressed Under Heart Failure Conditions
ImCa1 and ImCa2 could be detected in mitoplasts from both nonfailing and failing human hearts (Figure 1C and 1D). Notably, under failing heart conditions, the single-channel activity and gating of mCa1 and mCa2 were reduced, particularly exhibiting a lower open probability, a longer mean closed time, and a prolonged mean first latency, compared with nonfailing mitochondrial Ca2+ channels (Figures 1C, 2A, and 2
C; supplemental Figure I; and the Table). Moreover, we obtained a decrease in Ca2+ permeation strength and Ca2+ permeability of both Ca2+ channels under failing conditions, whereas the Ca2+ flux rate was unchanged (the Table).
Kinetics of Human Mitochondrial Ca2+ Uptake
Because the kinetics of mitochondrial Ca2+ uptake are currently a matter of intense debate,2,6,17 we analyzed the cumulative first latencies (time of first activation [
1] and steady-state activation [2]) and found that under nonfailing conditions the Ca2+ uptake via mCa1 and mCa2 channels is rapid, whereas in mitoplasts from failing hearts, a slower first activation and steady-state activation were evident (Figure 2D), consistent with reports from >30 years ago that mitochondrial Ca2+ uptake in failing hearts is decreased compared with control conditions.24,25
Subconductance Analysis
For ImCa2, only 1 full-open state was apparent. Conversely, ImCa1 is composed of subconductance levels (Figures 1C, 1D, and 4
). The maximum of simultaneously open channels (PMAX) for level 1 always exceeded 20%; for all other levels, PMAX was below 1%. Given the low stochastic probabilities for simultaneous openings of n independent channels (see Pn values in the legend of Figure 4E), the visual impression that ImCa1 openings are based on multilevels and not on multiple channels is verified by both PMAX and Pn.22 Thus, the different open levels of mCa1 are clearly from 1 single channel and are not caused by multiple openings of >1 channel (supplemental Table III). Notably, ImCa1 from nonfailing hearts exhibited 3 sublevels, which were increased to 5 sublevels under failing heart conditions, suggesting impaired opening of mCa1 channels in heart failure.
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Ca2+ Selectivity of Both Mitochondrial Ca2+ Channels
The main intracellular monovalent cation, ie, potassium, does not seem to contribute to ImCa1 or ImCa2 in the presence of Ca2+. The conductances of mCa1 and mCa2 increase with increasing [Ca2+]pipette (gmCa1: 7.1±0.43 pS [20 mmol/L, n=3], 9.8±0.89 pS [52.5 mmol/L, n=4], 10.9±0.33 pS [105 mmol/L, n=6]; gmCa2: 3.9±0.52 pS [20 mmol/L, n=3], 6.08±0.42 pS [52.5 mmol/L, n=3], 6.56±0.74 pS [105 mmol/L, n=5]), revealing a half-saturating concentration, Km, of 15.1±0.03 mmol/L Ca2+ (n=3 to 6) and 19.6±0.20 mmol/L Ca2+ (n=3 to 5; P<0.05) for mCa1 and mCa2, respectively (Figure 3B and 3C). The single-channel conductance of both channels in the presence of K+ at the cytoplasmic surface (pipette solution: 105 mmol/L K+, 52.5 mmol/L Ca2+) was not significantly changed compared with Ca2+ (52.5 mmol/L) alone [failing: gmCa1 (K+-Ca2+), 10.3±1.15 pS [n=3; P=NS versus gmCa1 (Ca2+)]; gmCa2 (K+-Ca2+), 5.80±0.62 pS [n=3; P=NS versus gmCa2 (Ca2+)]] (Figure 3B and 3D). Furthermore, the reversal potential was unaffected by extramitochondrial K+ [Vrev of ImCa1 (K+-Ca2+), 21.2±1.24 mV (n=3) versus Vrev of ImCa1 (52.5 mmol/L Ca2+), 21.0±0.88 mV (n=4; P=NS); Vrev of ImCa2 (K+-Ca2+), 10.6±1.09 mV (n=3) versus Vrev of ImCa2 (52.5 mmol/L Ca2+), 9.20±1.50 mV (n=3; P=NS)] (Figure 3B and 3D). These data support that mCa1 and mCa2 channels are less permeant or nonpermeant for K+ in the presence of Ca2+, thus preventing mitochondrial depolarization by these abundant cations.15
To rule out that the mitochondrial Ca2+ channels mCa1 and mCa2 conduct anions, we performed experiments with 105 mmol/L Ca2+-glutamate in the pipette solution (Figures 3A and 5D). Under these conditions, we observed the same channel behaviors and single-channel conductances as in the presence of Cl–, ie, with 105 mmol/L CaCl2 in the pipette solution [failing: gmCa1(Ca-glutamat), 10.1±0.29 pS (n=4; P=NS versus gmCa1(CaCl2)); gmCa2(Ca-glutamat), 5.92±0.20 pS (n=4; P<0.05 versus gmCa1(Ca-glutamat); P=NS versus gmCa2(CaCl2))], supporting that mCa1 and mCa2 were not conducting Cl–.
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Pharmacological Characteristics of Both Mitochondrial Ca2+ Channels
Reliably evaluating the effect of modulators channel stability over time is important. Under control conditions, mCa1 and mCa2 were stable over a recording time of 20 minutes (no rundown; Figure 5A). Similar to the mitochondrial Ca2+ uptake via the MCU,2,15,17 ImCa1 was inhibited by 200 nmol/L Ru360 (Figure 5B and 5C). ImCa2 was unaffected by 200 nmol/L Ru360 but was significantly suppressed by 10 µmol/L Ru360 (Figure 5B and 5C), consistent with reports suggesting an RuR-insensitive/low-sensitivity mitochondrial Ca2+ uptake in addition to the classic RuR-sensitive MCU.2,12,16 These data were derived from mitoplasts under nonfailing heart conditions. Pharmacological experiments of mitoplasts from failing hearts showed a more pronounced Ru360 inhibition (Figure 5D and 5E).
To further support the identity of mCa1 and mCa2, we performed additional agonist experiments. Spermine (1 mmol/L), an activator of the MCU,26 significantly increased both channel activities that were blocked—after washout—by 10 µmol/L Ru360 (Figure 5D and 5E). The pharmacological effects were stronger for mCa1 than for mCa2, supporting that mCa1 is the RuR-sensitive MCU.
Although the remaining Ca2+ uptake in the presence of nanomolar concentrations of Ru360 might be due to alternative Ca2+ pathways via the mitochondrial Na+-Ca2+ exchanger12,27 or mitochondrial ryanodine receptor,28 addition of CGP-37157 (10 µmol/L), cyclosporin A (10 µmol/L), dantrolene (10 µmol/L), S(-)-BayK 8644 (10 µmol/L), or xestospongin C (10 µmol/L) to the bath or pipette solution did not affect ImCa1 or ImCa2 (supplemental Table IV), indicating that these 2 Ca2+ currents were not mediated by the mitochondrial Na+-Ca2+ exchanger,29 mitochondrial permeability transition pore,30 mitochondrial ryanodine receptor,28 mitochondrial dihydropyridine site,31 or an IP3 receptor,32 respectively.
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Discussion |
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The present study demonstrates that 2 distinct ion channels underlie human mitochondrial Ca2+ sequestration. Both channels are voltage gated, are Ca2+ selective in the presence of the predominant intracellular monovalent cation K+ and the anion Cl–, and exhibit increased activity at more negative potentials, making them particularly sufficient for Ca2+ uptake driven by the large electrochemical gradient for Ca2+ across the inner membrane of energized mitochondria. mCa1 and mCa2 channels can clearly be distinguished by their unique single-channel characteristics and different sensitivity to Ru360, explaining previous reports of mitochondrial Ca2+ uptake via the MCU and a non-MCU pathway.2,12,17
Human mCa1 channels share some properties with MiCa channels recorded in COS-7 cells, ie, a similar range of single-channel conductance, amplitude, and Ca2+ flux, an increased open probability at more negative voltages, subconductance states, and sensitivity to nanomolar Ru360 concentrations, but differ markedly in gating parameters like open and closed times, suggesting species- and/or tissue-specific diversity of the MCU.15 Although suppression of the so-called non-MCU–type, RuR-insensitive Ca2+ uptake in mammalian mitochondria by high RuR concentrations led to the assumption that it might be mediated by a MCU with reduced RuR sensitivity,2,12,17 distinct single-channel properties of ImCa1 and ImCa2, even when recorded simultaneously in 1 mitoplast, indicate that these 2 channels are composed of different protein complexes. Previously, a higher Ru360 sensitivity has been obtained for mitochondrial Ca2+ uptake linked to the MCU with complete suppression by a concentration of 200 nmol/L.2,12–15 However, we do not consider these and our results contradictory because the difference in inhibition may be due, at least partly, to different experimental conditions, given that for other channels and compounds, a lower sensitivity in cell-attached versus whole-cell configuration also has been reported.33,34 Moreover, activation of both mCa1 and mCa2 by spermine further supports their Ca2+ channel identity of the MCU and non-MCU type.26
Although mitochondrial dysfunction in disease and aging has been related mainly to direct alterations of the respiratory chain or 
,35,36 here we demonstrate reduced function of mitochondrial Ca2+-channels and thus of an indirect modulator of energy and reactive oxygen species production in a disease known to be associated with profound changes in intracellular Ca2+ homeostasis and metabolism.18 Alterations of the mitochondrial matrix composition (ie, Ca2+ buffering/alkalization, matrix-free [Ca2+], H+ translocation, protein expression) might also have contributed to the observed differences in mCa1/2 channel properties of mitochondria from failing and nonfailing hearts in the present study. Moreover, a depolarization of the potential across the inner mitochondrial membrane, which has been reported for cardiomyopathic hamsters, might have decreased the driving force and thus Ca2+ channel activity under failing heart conditions.36 However, in addition to differences in the basal biophysical channel behavior, we obtained marked differences in the pharmacological behavior under nonfailing (Figure 5B and 5C) and failing (Figure 5D and 5E) conditions, supporting the hypothesis of (additional) changes in channel structure, subunit composition, or binding sites in diseased state. In this respect, a possible contribution of altered expression levels of uncoupling proteins 2/3, which recently have been implicated in the function of the MCU, has to be determined.12,37,38
Maintaining a high ATP supply is critically important for maintaining cardiac performance. The daily turnover of ATP in the heart is very many times that of the myocardial ATP pool. Thus, even subtle variations in the efficiency of energy generation and use may have a cumulative impact on cellular energy levels. Numerous studies have identified decreased cardiac energy levels and flux as consistent features of heart failure.39,40 Analysis of human biopsy specimens demonstrated that ATP is 25% to 30% lower in failing human hearts compared with controls.41,42 Although the myocardial tissue is well oxygenized, it remains to be determined whether the chronic, progressive loss of ATP in heart failure occurs because de novo synthesis of ATP is slowed or fails or because ATP degradation is accelerated. Our observations of suppressed mitochondrial Ca2+ channel activity support the notion that this mismatch might be due, at least partly, to a reduced mitochondrial Ca2+ uptake and thus Ca2+-induced ATP generation.3,43 Only a few reports are contrary to this dogma that mitochondrial Ca2+ accumulation accelerates mitochondrial metabolism.44,45 In these experiments, conditions are present in which the depolarizing effect of Ca2+ on the inner mitochondrial membrane exceeds its stimulatory effect on the respiratory chain and dehydrogenases and consequently leads to a Ca2+-induced decrease in NADH levels. In such a scenario, reduced mitochondrial Ca2+ channel activity would prevent further mitochondrial Ca2+ overload and thus decline in ATP synthesis.
Furthermore, mitochondrial Ca2+ uptake exhibits dual contribution in the regulation and control of reactive oxygen species production, which has been implicated in triggering mitochondrial DNA mutations and cell death.46 Mitochondrial Ca2+ sequestration has been reported to facilitate the generation of reactive oxygen species within mitochondria,47 whereas dissipating on excessive Ca2+ accumulation is expected to counteract generation of · O2– during oxidative phosphorylation.48,49 Therefore, it remains unclear whether suppressed activity of mCa1 and mCa2 in human heart failure should be considered adaptive, maladaptive, or a cause of progressive decline in pump function.
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Conclusions |
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TopAbstractIntroductionMethodsResultsDiscussionConclusionsReferences |
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This study demonstrates for the first time that human cardiac mitochondrial Ca2+ uptake is regulated by 2 different selective mitochondrial Ca2+ channels: mCa1, the RuR-sensitive Ca2+ uniporter, and mCa2, an RuR-insensitive/low-sensitivity non-MCU–type mitochondrial Ca2+ channel. Furthermore, we found that both mitochondrial Ca2+ currents are less active under failing heart conditions than in mitoplasts from nonfailing hearts.
On the basis of our experiments, we conclude that the human MCU and the RuR-insensitive Ca2+ uptake pathway are the Ca2+-selective ion channels mCa1 and mCa2, respectively, which are functionally impaired in the diseased heart state.
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Acknowledgments |
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We thank Nadine Henn and Iris Berg for technical assistance.
Sources of Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (to Dr Hoppe), Marga and Walter Boll-Stiftung (to Dr Hoppe), and Köln Fortune (to Dr Hoppe).
Disclosures
None.
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References |
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The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.108.835389/DC1.
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