Energetic Crosstalk Between Organelles
Architectural Integration of Energy Production and Utilization
Cells with high and fluctuating energy demands such as cardiomyocytes need efficient systems to link energy production to energy utilization. This is achieved in part by compartmentalized energy transfer enzymes such as creatine kinase (CK). However, hearts from CK-deficient mice develop normal cardiac function under conditions of moderate workload. We have therefore investigated whether a direct functional interplay exists between mitochondria and sarcoplasmic reticulum or between mitochondria and myofilaments in cardiac cells that catalyzes direct energy and signal channeling between organelles. We used the selective permeabilization of sarcolemmal membranes with saponin to study the functional interactions between organelles within the cellular architecture. We measured contractile kinetics, oxygen consumption, and caffeine-induced tension transients. The results show that in hearts of normal mice, ATP produced by mitochondria (supplied with substrates, oxygen, and adenine nucleotides) was able to sustain calcium uptake and contractile speed. Moreover, direct mitochondrially supplied ATP was nearly as effective as CK-supplied ATP and much more effective than externally supplied ATP, suggesting that a direct ATP/ADP channeling exists between the sites of energy production (mitochondria) and energy utilization (sarcoplasmic reticulum and myofilaments). On the other hand, in cardiac cells of mice deficient in mitochondrial and cytosolic CK, marked cytoarchitectural modifications were observed, and direct adenine nucleotide channeling between mitochondria and organelles was still effective for sarcoplasmic reticulum and myofilaments. Such direct crosstalk between organelles may explain the preserved cardiac function of CK-deficient mice under moderate workloads.
Differentiation and maturation of adult mammalian muscle cells lead to complex specialization and organization. In cardiac cells, specialized cellular functions are highly organized within structural and functional compartments. Energy-consuming processes are localized to the sarcoplasmic reticulum (SR) and myofibrillar compartments, while energy production occurs mainly within mitochondria. Muscle cells contain complex and specialized energy transfer systems, which efficiently link energy production and utilization. One such system is the family of creatine kinase (CK) isoenzymes that catalyze the reversible transfer of a phosphate moiety between creatine (Cr) and ATP. The mitochondrial sarcomeric isoenzyme (mi-CK) is bound to the outer surface of the inner mitochondrial membrane so that ATP generated by oxidative phosphorylation is transphosphorylated to phosphocreatine (PCr).1–3 On the other hand, the cytosolic isoenzyme (MM-CK) that is structurally associated with myofibrils and SR membranes can use PCr to rephosphorylate all of the ADP produced by the ATPases and thus provide enough energy for normal contractile kinetics or SR calcium uptake.4–7
Recent studies have revealed that mice lacking one or both of the MM-CK and mi-CK isoforms (CK−/−) are viable and develop nearly normal cardiac function under the conditions of moderate workload.8–11 This suggests that other mechanisms may ensure efficient energy transfer and signal transduction between sites of energy production and energy utilization. Indeed, increases in mitochondrial volume and cytoarchitectural rearrangements have been observed in CK−/− mice10 suggesting adaptational mechanisms to CK deficiency. One possibility might be that a direct functional interplay between subcellular organelles exists that catalyzes direct energy and signal channeling between mitochondria and the SR on the one hand and between mitochondria and myofilaments on the other. Indeed, mitochondria appear to be clustered at sites of high ATP demand and are organized into highly ordered elongated bundles, wrapped around the myofibrils and in contact with the SR.12 Structural contacts between the SR and mitochondria have been revealed by electron microscopy,13 and compelling evidence points to a coordination between these organelles at the level of calcium homeostasis14–16 and regulation of ATP production.17 Previous studies have suggested a possible direct functional interaction between these ATP-producing and -consuming intracellular organelles at the level of energy transfer.18–20
Functional studies using skinned fibers provide a unique mean to investigate these possible interactions. Indeed, the use of specific membrane permeabilization with detergents allows for the study of organelle function while maintaining the cellular architecture and controlling the intracellular milieu. This experimental approach is a valuable tool for studying mitochondrial function and regulation in situ and can demonstrate the functional coupling between bound MM-CK and cardiac sarcoplasmic reticulum (SERC-ATPase) or myofibrillar ATPase.4,6,21 In the present study, using the selective permeabilization of cardiac sarcolemmal membranes with saponin, we have investigated (1) whether direct ATP supply by mitochondria can provide energy to the SERC-ATPase for calcium uptake or to myosin-ATPase for contraction, (2) whether ATP supplied directly by mitochondria is as effective as ATP supplied by bound CK or as ATP supplied from the surrounding medium, and (3) how ATP is supplied to energy-utilizing organelles in cardiac cells of mice lacking sarcomeric mitochondrial and cytosolic CKs (CK−/− mice).
Materials and Methods
Preparation of Skinned Fibers and Solutions
Procedures involved in the generation and genotyping of mitochondrial/MM-CK null mice have been described in detail elsewhere.10 Three-month-old male control C57BL/6 and CK−/− mice were anesthetized with an intraperitoneal injection of sodium thiopental according to the recommendations of the Institutional Animal Care Committee (INSERM, Paris, France). The hearts were removed and rinsed in an ice-cold Ca2+-free Krebs solution equilibrated with 95%O2/5% CO2. Fibers (diameter 150 to 250 μm) were dissected from left ventricular papillary muscles. Specific permeabilization of sarcolemma was obtained by incubating the fibers for 30 minutes in relaxing solution (basic solution at pCa 9, see below) containing additionally 50 μg/mL saponin in the presence of 5 μg/mL leupeptin at 4°C. After skinning, fibers were kept in the relaxing solution at 4°C until further use. Solutions for permeabilized fibers were calculated using the computer program of Fabiato.22 All solutions were prepared using the basic solution. This solution contained (in mmol/L) ethylene glycol-bis(β-aminoethyl ether)N,N,N′,N′-tetra-acetic acid 10 (EGTA; except for prerelease and release solutions, 0.2), N,N-bis[2-hydroxyethyl]-2-aminoethanesulfonic acid 60 (BES, pH 7.1), free Mg2+ 1, taurine 20, glutamic acid 5, malic acid 2, K2HPO4 3, dithiothreitol 0.5, P1,P5 diadenosine pentaphosphate 0.040 (to inhibit adenylate kinase activity), and MgATP 3.16 (except otherwise stated); ionic strength was adjusted to 160 mmol/L with potassium methanesulfonate. Desired pCa was obtained by varying CaK2EGTA/K2EGTA ratio.
Electron Microscopy Study
Samples of the left ventricles, taken from 5 hearts of wild-type or CK−/− mice, were washed with Ca2+-free Krebs solution for 10 minutes and fixed with 2% glutaraldehyde. After fixation, tissue samples were postfixed with 1% osmium tetroxide, contrasted with 1% uranyl acetate in ethanol, dehydrated, and embedded in Durcupan (Fluka Chemie AG). Ultrathin longitudinal sections were stained with lead citrate and studied using a JEM 1200 (JEOL) electron microscope.
Estimation of SR Ca2+ Uptake
The experimental protocol used in this study was a modified version of that described by Minajeva et al4 (for more detail, see the online data supplement available at http://www.circresaha.org). After emptying the SR by a brief application of caffeine (5 mmol/L), SR loading was carried out in solutions with different ATP sources at pCa 6.5. These solutions contained in addition to basic solution (in mmol/L) ADP 1, instead of ATP for ADP+MITO solution; azide 2, to inhibit mitochondria for ATP solution or no azide for MITO solution; PCr 12 and azide 2, for ATP+PCr solution; or no azide for ATP+PCr+MITO solution. In some experiments, mitochondrial substrates (glutamate and malate) were omitted or oligomycin (40 μmol/L), an inhibitor of mitochondrial ATPase activity, was added. Calcium release was induced by 5 mmol/L caffeine in the presence of PCr and ATP for control mice or with ATP alone when comparing control and CK−/− mice (see below), to ensure comparable conditions of myofilament activation. Tension at peak and tension-time integral were measured and analyzed as previously described.4
Myofibrillar crossbridge cycling rate, which is the functional counterpart of actomyosin ATPase activity, was estimated by the quick length-change technique as previously described23 (for more detail, see online data supplement).
Oxygen Consumption and Biochemical Determinations
Respiratory rates were determined using a Clark electrode (Strathkelvin Instruments) as described previously24 (for more detail, see online data supplement). For estimating the competition between mitochondria and CK for ATP supply to ATPases, fibers (≈0.35 mg of dry weight) were transferred into 1 mL oxygraphic cell–containing basic solution at pCa 6.5 and the respiratory rate was determined. Five minutes later, 12 mmol/L PCr was added to the chamber and oxygen measurements continued for an additional 5 minutes. Thereafter, a mixture of atractyloside (20 μmol/L) and oligomycin (20 μmol/L) was added to quickly stop mitochondrial respiration and measurements continued for an additional 5 minutes. During all these steps, samples from the respiration media were collected and the Cr concentration was determined.
Values are expressed as mean±SE. A Student’s t test was used to determine the statistical difference of means between control and CK−/− groups. Within a group, statistical significance of differences between the averages was estimated by a repeated-measures of ANOVA using Dunnett’s post hoc test.
An expanded Materials and Methods section can be found in the online data supplement available at http://www.circresaha.org.
Mitochondria-Supported SR Ca2+ Load
We previously reported that in saponin-permeabilized fibers, the SR could be loaded much more efficiently with PCr and ATP compared with ATP alone. We concluded that CK bound to the SR membrane was able to provide ATP and withdraw ADP close to the Ca2+-ATPase-driven pump.4,25
We wondered whether a functional compartmentation can occur in normal hearts between the SR and mitochondria, two organelles known to establish physical contacts. Fibers were incubated in a solution containing mitochondrial substrates and ADP (ADP+MITO solution) at pCa 6.5 for 5 minutes, to load the SR at the expense of mitochondrially produced energy. When sequestered calcium was released with caffeine, a tension transient could be elicited, showing that SR has been effectively loaded with calcium (Figure 1, left). This uptake was time-dependent as it increased with longer incubation periods (results not shown). Sodium azide, an inhibitor of mitochondrial respiration, completely abolished mitochondrially supported SR loading. Thus, mitochondrially produced energy could time dependently support SR Ca2+ loading. However, these experiments were unable to answer the question of the efficacy of the mitochondrially supported SR load, because high ADP concentrations are known to inhibit or reverse the SR calcium pump. We thus replaced external ADP with ATP in the loading solutions. Under these conditions, mitochondria can only use the ADP coming from the hydrolysis of ATP catalyzed by the cellular ATPases.1 SR load was up to 4-fold more effective when ATP was used instead of ADP (Figure 1, right). Moreover, when mitochondria were blocked with 2 mmol/L sodium azide, only 5% of the response remained, suggesting that most of the ATP used was of mitochondrial origin and not from external ATP. Thus, in normal hearts, mitochondrially produced ATP appeared far more effective in supporting SR calcium load than exogenous ATP, showing that mitochondria can effectively maintain high ATP/ADP ratio near the SR calcium pump.
Our next step was to compare the efficacy of mitochondrially produced ATP with the efficacy of ATP regenerated by bound CK. For this, we compared the SR calcium release after 5 minutes of loading at pCa 6.5 supported either (1) by external ATP, (2) by external ATP and mitochondria, (3) by external ATP and PCr, or (4) by external ATP, PCr, and mitochondria. Either in the presence of active mitochondria or PCr, or both, the SR load was significantly higher than it was with external ATP alone (Figure 2). We checked whether azide could induce an increase in mitochondrial ATPase activity, leading to local ATP depletion. However, when mitochondria were blocked by omitting substrates, or by azide, or by azide with oligomycin, an inhibitor of mitochondrial ATPase activity, tension transients were much lower than when SR was loaded with mitochondria (Figure 3A). In the following experiments, azide was preferred because of simplicity of use and high reversibility, which allowed the loading conditions to be randomized. Averaged peak transients and tension-time integrals were much lower when external ATP was supplied than when ATP was produced by either CK or mitochondria (see online data supplement available at http://www.circresaha.org). Normalizing tension-time integrals to the ATP+MITO condition for each fiber showed that the ATP-supported load was almost 30 times less effective than other loading conditions (Figure 3C). As in ATP+MITO, the tension-time integral increased linearly for up to 15 minutes of loading (results not shown); SR was not saturated at 5 minutes of loading for any conditions. Thus, mitochondria can support SR calcium load almost as effectively as the PCr/CK system. Moreover, each loading condition seems to be maximally efficient, because loading in ATP+PCr+MITO solution was not significantly higher than in ATP+MITO.
Mitochondria-Supported Myosin ATPase Activity
In the next experiments, we checked whether mitochondrially supplied ATP could also be effectively used for myofibrillar function. It has been previously shown that bound CK is necessary for optimal myosin ATPase activity and crossbridge cycling.26 Functional activity of myofibrils was estimated by measuring the rate constant of tension changes after quick changes in length33 under the different ATP supply conditions as used for the SR. Figure 3B shows that whatever the conditions, when mitochondria were inhibited, crossbridge cycling rate was much lower than when ATP was produced by mitochondria, showing again that the low efficacy of external ATP was not due to activation of mitochondrial ATPase. Results with different loading conditions are presented in Figure 3D as relative values (absolute values provided in the online data supplement). The rate constant increased significantly in the presence of active mitochondria or PCr when compared with external ATP only. In the presence of active mitochondria and PCr, the rate constant was even higher than with active mitochondria alone. The results demonstrate that in myofibrils, as in SR of normal cardiac cells, mitochondria can favor the ATP/ADP compartmentation nearly as effectively as PCr.
Competition Between Mitochondria and CK
From the above experiments, it was clear that mitochondrially produced energy alone was sufficient to support SR Ca2+ load and myosin-ATPase activity to almost the same extent as bound CK. However, it remained unclear which mechanism of ATP regeneration dominates when both mechanisms are working together. We therefore simultaneously estimated the activity of the CK reaction by measuring Cr formation and the mitochondrial ATP synthesis rate by measuring ADP-dependent oxygen consumption (Figure 4). When both CK and mitochondria were working, the rate of Cr formation amounted to 53±3 nmol Cr · min−1 · mg dry weight−1 whereas oxygen consumption rate was 5.3±1.2 nmol O2 · min−1 · mg dry weight−1. Assuming a one-to-one ATP production per Cr and an ADP/O ratio of ≤3, we estimated that under our conditions ≥65±4% of the ATP consumed came from the CK reaction and ≤35±4% from mitochondria. However, blocking the CK reaction by eliminating PCr led to an 80% compensatory activation of mitochondria to 9.7±1.5 nmol O2 · min−1 · mg dry weight−1 (P<0.01). Conversely, inhibiting mitochondria induced a 40% increase in Cr production to 77±9 nmol · min−1 · mg dry weight−1 (P<0.05). Thus, both the mitochondria and CK system are working on a competitive basis and can compensate for each other.
Mitochondrial Function and Cytoarchitecture in CK−/− Mice
Our experiments suggest that in permeabilized fibers of normal animals, mitochondria can maintain a high ATP/ADP ratio near the cellular ATPases when CK is not functionally active. We have further tested this in mice deficient in cytosolic and mitochondrial CKs (CK−/−). These mice exhibited a slight cardiac hypertrophy (heart weight per body weight ratio 6.0±0.3 versus 5.0±0.1 mg · g−1 in control mice, P<0.05). As expected, CK activity was very low in CK−/− cardiac tissue (3.7±0.7 versus 480±45 IU · g wet weight−1 in control). There was no overexpression of citrate synthase, a marker of mitochondrial content (146±36 and 144±18 IU · g wet weight−1) or adenylate kinase, another ATP-regenerating enzyme (178±19 and 189±23 IU · g wet weight−1) in control versus CK−/− mice, respectively. Moreover, basal and maximal respiration rates as well as acceptor control ratio did not differ between control and CK−/− mice (Table). However, mitochondria from CK−/− mice exhibited a higher sensitivity to external ADP than control. As expected, Cr decreased the Km for ADP in control mice due to the functional coupling between translocase and mitochondrial CK, whereas such an effect was absent in CK−/− mice.
Examination of the ultrastructure of cardiac fibers showed that although in wild-type mice mitochondria are arranged in longitudinally running columns between strips of contractile proteins (Figure 5, left), in CK−/− mice, mitochondria and myofilaments show very obvious signs of reorganization. Frequent splitting of myofibrils resulted in formation of thinner myofilament bundles and their deviation from the longitudinal direction (Figure 5, middle). Moreover, the sarcomere structure appeared altered with decreased A-bands. Abundant mitochondria form bulk regions and fill all the space between myofibrils. In some places, mitochondria entering the myofibrils at sites of splitting could be observed (Figure 5, right).
Our next step was to investigate whether mitochondrial ATP could maintain calcium uptake and crossbridge cycling rate in ventricular fibers of CK−/− mice. In these experiments all steps were performed in the absence of PCr to create equal conditions for control and CK−/− fibers. The results demonstrate that mitochondria-supported calcium load was similar in both groups (Figure 6A). Similarly, the rate constant of crossbridge cycling in ATP+MITO solution did not significantly differ from control mice (107±11 versus 132±11 s−1 in control; Figure 6B), being largely faster than with ATP alone (38±7 s−1). However, this last value was significantly lower in CK−/− than in control mice (63±4 s−1, P<0.02). Thus, in CK-deficient animals, externally added ATP poorly supported the SR- and myosin-ATPases, whereas mitochondria could support SR calcium load and crossbridge cycling rate to a similar extent as in control mice.
Functional microcompartmentation of intracellular metabolites and substrates due to the spatial distribution of mitochondria and glycolytic complexes has long been recognized.18–20 In this respect, striated muscle cells represent a paradigm of spatial organization between energy-producing and energy-utilizing sites, because muscle cell organization should match the high and fluctuating energy utilization needed for contractile work. Mitochondria are the main providers of energy in oxidative muscle and heart, and previous studies18–20 have suggested a direct interaction between mitochondria and the main ATP-consuming compartments, the SR, and myofibrils. In digitonin-lysed cardiomyocytes, Altschuld et al27 reported that Ca45 uptake in the presence of ATP was not sensitive to mitochondrial inhibitors, although this possibility was not directly addressed in their study.
We took advantage of the cell permeabilization technique to investigate such a possibility.6,21 The present results show that direct mitochondrial ATP supply and/or ADP withdrawal can support the kinetic and thermodynamic requirements of both the myosin ATPase and SERC-ATPase. This mitochondrial ATP supply appears nearly as effective as CK-supplied ATP and is much more effective than externally supplied ATP, in sustaining calcium uptake and contractile speed.
Direct Channeling of ATP and ADP Between Organelles
These results can be explained in view of the highly ordered and densely packed organization of adult mammalian cardiac cells. Close proximity of mitochondria, SR, and myofilaments leads to a situation where nucleotide transfer can occur much more freely between the organelles than to or from the cytoplasm. The direct channeling of ATP and ADP between mitochondria- and ATP-utilizing structures such as the SR and myofilaments establishes a direct crosstalk between organelles through compartmentation of adenine nucleotides. Indeed, it has been demonstrated that access of external ADP to the mitochondrial matrix is greatly impaired in oxidative muscles (with an apparent Km for ADP largely exceeding its cytosolic level21), whereas internally produced ADP has preferential access to mitochondria.28 It needs to be determined whether the efficacy results primarily from facilitation of ADP withdrawal or ATP supply. Skinned fiber studies, together with recent mathematical modeling have pointed out that ADP diffusion is indeed limited in cardiac cells, suggesting that this limited diffusion is the basis of adenine nucleotide compartmentation.6,21,29 As a result of the low Km of the ATPases for ATP and the high Ki for ADP, it is probable that ADP accumulation is the limiting factor for ATPase activity. Myosin ATPase and SERC-ATPase are two highly regulated enzymes, whose effective regulation depends on the relief of substrate limitation and product accumulation. Moreover, SR calcium ATPase functions in both forward and reverse mode, and its efficiency is directly dependent on ΔGATP.30 Indeed, accumulation of ADP near the active sites slows down myosin ATPase and crossbridge cycling rate (see Ventura-Clapier et al6 for review) or impairs SERC-ATPase activity and calcium uptake.4 Both phosphotransfer kinases and mitochondria, by sharing their products and their substrates with the ATPases are able to locally control ATP and ADP concentrations, thus exerting a thermodynamic and kinetic control over these enzymes.
The corollary of these observations is that there is no reason to expect a direct correlation between cardiac work and global ADP concentration as inferred from nuclear magnetic resonance (NMR) experiments. This is emphasized by the fact that changes in workload and increases in metabolic rates can proceed in heart muscle without marked changes in PCr and adenine nucleotide contents,9,31 supporting the alternative view that compartmentation and high-energy phosphoryl transfer through phosphotransferases regulate metabolic rates. Recently, Joubert et al32 demonstrated that the discrepancy between forward and reverse CK fluxes observed in NMR experiments could be explained by a pool of ATP not participating in the CK reaction. This pool of ATP representing 20% of total cellular ATP could represent that part of ATP directly channeled from mitochondria to ATPases.
Bound CK and Mitochondria in Controlling Local ATP/ADP Pools
Our results additionally demonstrated that ATP supplied directly from mitochondria could be nearly as effective as ATP supplied by CK/PCr. Moreover, inhibiting one of these mechanisms led to the immediate activation of the other. The vital importance of the local control of the adenylate pool is underscored by the existence of multiple coexisting systems. However, all the systems may not be exactly equivalent, although they appear to cooperate within the cell. The CK system appeared more effective than the crosstalk between organelles because, when both systems were active, two thirds of the energy production came from bound CKs. In intact cells, the CK shuttle could be even more important because the high cytosolic content of CK could take part in local ATP regeneration. Moreover, as a result of the near-equilibrium nature of the cytosolic CK reaction, it would be able to spread the energy signal all over the cell, thus ensuring coordination of the different cellular substructures, whereas organelle crosstalk would be more spatially restricted. This sheds light on the observations obtained in rabbit heart that CK inhibition accelerates the response time of mitochondria during rapid workload steps.33 Indeed, inhibiting CK would increase local ADP for mitochondria thus favoring the direct interorganelle crosstalk, resulting in more rapid mitochondrial stimulation. On the other hand, direct interaction between mitochondria and SR or myofilaments could be the physical basis to explain metabolic waves and spots observed in energy-depleted isolated cardiac cells.34
In fact, the picture may be even more complex because glycolytic complexes are also associated with myofibrils and SR and that at least for SR, glycolytic complexes efficiently participate in energy supply.35,36 Moreover, adenylate kinase, which is also bound to intracellular organelles and within mitochondria, can also participate in phosphotransfer in cardiac cells, particularly when the CK reaction is impaired.37 Thus, highly structured cytoarchitecture involving direct organelle interaction, compartmentalized phosphotransfer kinases, and bound glycolytic enzymes allows high efficiency and fine-tuning of energy transduction systems and cardiac muscle function.
The partial redundancy of local ATP/ADP-controlling systems in cardiac muscle is well illustrated in CK knockout mice. It is recognized that CK deletions do not form a serious obstacle to normal heart function under laboratory conditions.38 Isolated hearts from CK−/− mice have comparable function at baseline and a nearly equal response to a challenging intervention than control mice,8,9 although at the moderate workload that could be achieved in Langendorff-perfused hearts.29 However, because local control of ATP/ADP close to cellular ATPases has a critical influence on enzyme activity, alternative ATP/ADP control systems and compensatory mechanisms should be operating in CK−/− mouse heart to overcome diffusion limitation and to preserve cardiac function at least at moderate levels of activity. Mitochondrial content, either morphologically, biochemically, or functionally (the present study) determined, was not altered in CK−/− hear, although it was greatly increased in skeletal muscles.10,38 However, as previously described in MM-CK null mice,24 the sensitivity of mitochondrial respiration to external ADP was increased in CK−/− mice and could partially compensate for the lack of mitochondrial CK, by allowing the cytosolic ADP signal to directly reach the mitochondrial matrix in the absence of channeling through CK. Moreover, we observed a remarkable reorganization at the cytoarchitectural level in the hearts of CK−/− mice. Mitochondria reorganized within myofilaments and tended to decrease diffusion distances, showing that subcellular organization is sensitive to energy deficiency. Cell remodeling, direct crosstalk between organelles, and increased mitochondrial ADP sensitivity can obviously participate in such compensatory mechanisms. In addition, we have recently reported that glycolytic enzymes can also participate in SR calcium uptake efficiency but not for myofibrillar function.35 Although adenylate kinase was not upregulated in CK−/− mice, it could also contribute to intracellular energy fluxes as a compensatory mechanism when CK is inhibited.37 The presence of alternative systems taking part in facilitated ADP diffusion partially explains the mild phenotype observed in these mice, at least under normal laboratory conditions. However, it might be anticipated that, although these systems may appear redundant at rest or during moderate workload in the heart, it is highly probable that at a higher energy demand, the different systems able to control local adenylate pools would have to be additively recruited.
This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM) grants (to M.N., A.K., and E.B.) and a Federation of European Biochemical Societies (FEBS) grant (to A.M.). R.V.-C. is supported by “Center National de la Recherche Scientifique.” We gratefully acknowledge B. Wieringa and F. Oerlemans for providing the engineered mice. R. Fischmeister is acknowledged for continuous support.
Original received November 27, 2000; revision received May 23, 2001; accepted May 23, 2001.
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