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International audience; AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis under conditions of energy stress. Though heart is one of the most energy requiring organs and depends on a perfect match of energy supply with high and fluctuating energy demand to maintain its contractile performance, the role of AMPK in this organ is still not entirely clear, in particular in a non-pathological setting. In this work, we characterized cardiomyocyte-specific, inducible AMPKα1 and α2 knockout mice (KO), where KO was induced at the age of 8 weeks, and assessed their phenotype under physiological conditions. In the heart of KO mice, both AMPKα isoforms were strongly reduced and thus deleted in a large part of cardiomyocytes already 2 weeks after tamoxifen administration, persisting during the entire study period. AMPK KO had no effect on heart function at baseline, but alterations were observed under increased workload induced by dobutamine stress, consistent with lower endurance exercise capacity observed in AMPK KO mice. AMPKα deletion also induced a decrease in basal metabolic rate (oxygen uptake, energy expenditure) together with a trend to lower locomotor activity of AMPK KO mice 12 months after tamoxifen administration. Loss of AMPK resulted in multiple alterations of cardiac mitochondria: reduced respiration with complex I substrates as measured in isolated mitochondria, reduced activity of complexes I and IV, and a shift in mitochondrial cristae morphology from lamellar to mixed lamellar-tubular. A strong tendency to diminished ATP and glycogen level was observed in older animals, 1 year after tamoxifen administration. Our study suggests important roles of cardiac AMPK at increased cardiac workload, potentially limiting exercise performance. This is at least partially due to impaired mitochondrial function and bioenergetics which degrades with age.

Mitochondrial nucleoside diphosphate kinase (NDPK-D; synonyms: NME4, NM23-H4) represents the major mitochondrial NDP kinase. The homohexameric complex emerged as a protein with multiple functions in bioenergetics and phospholipid signaling. It occurs at different but precise mitochondrial locations and can affect among other mitochondrial shapes and dynamics, as well as the specific elimination of defective mitochondria or cells via mitophagy or apoptosis. With these various functions in cell homeostasis, NDPK-D/NME4 adds to the group of so-called moonlighting (or gene sharing) proteins.

International audience; Mitophagy is an emerging paradigm for mitochondrial quality control and cell homeostasis. Dysregulation of mitophagy can lead to human pathologies such as neurodegenerative disorders and contributes to the aging process. Complex protein signaling cascades have been described that regulate mitophagy. We have identified a novel lipid signaling pathway that involves the phospholipid cardiolipin (CL). CL is synthesized and normally confined at the inner mitochondrial membrane. However, upon a mitophagic trigger, ie, collapse of the inner membrane potential, CL is rapidly externalized to the mitochondrial surface with the assistance of the hexameric nucleoside diphosphate kinase D (NME4, NDPK-D, or NM23-H4). In addition to its NDP kinase activity, NME4/NDPK-D shows intermembrane phospholipid transfer activity in vitro and in cellular systems, which relies on NME4/NDPK-D interaction with CL, CL-dependent crosslinking of inner and outer mitochondrial membranes by symmetrical, hexameric NME4/NDPK-D, and a putative NME4/NDPK-D-based CL-transfer pathway. CL exposed at the mitochondrial surface then serves as an 'eat me' signal for the mitophagic machinery; it is recognized by the LC3 receptor of autophagosomes, targeting the dysfunctional mitochondrion to lysosomal degradation. Similar NME4-supported CL externalization is likely also involved in apoptosis and inflammatory reactions.

International audience; There is an increasing body of evidence for local circuits of ATP generation and consumption that are largely independent of global cellular ATP levels. These are mostly based on the formation of multiprotein(-lipid) complexes and diffusion limitations existing in cells at different levels of organization, e.g., due to the viscosity of the cytosolic medium, macromolecular crowding, multiple and bulky intracellular structures, or controlled permeability across membranes. Enzymes generating ATP or GTP are found associated with ATPases and GTPases enabling the direct fueling of these energy-dependent processes, and thereby implying that it is the local and not the global concentration of high-energy metabolites that is functionally relevant. A paradigm for such microcompartmentation is creatine kinase (CK). Cytosolic and mitochondrial isoforms of CK constitute a well established energy buffering and shuttling system whose functions are very much based on local association of CK isoforms with ATP-providing and ATP-consuming processes. Here we review current knowledge on the subcellular localization and direct protein and lipid interactions of CK isoforms, in particular about cytosolic brain-type CK (BCK) much less is known compared to muscle-type CK (MCK). We further present novel data on BCK, based on three different experimental approaches: (1) co-purification experiments, suggesting association of BCK with membrane structures such as synaptic vesicles and mitochondria, involving hydrophobic and electrostatic interactions, respectively; (2) yeast-two-hybrid analysis using cytosolic split-protein assays and the identifying membrane proteins VAMP2, VAMP3 and JWA as putative BCK interaction partners; and (3) phosphorylation experiments, showing that the cellular energy sensor AMP-activated protein kinase (AMPK) is able to phosphorylate BCK at serine 6 to trigger BCK localization at the ER, in close vicinity of the highly energy-demanding Ca(2+) ATPase pump. Thus, membrane localization of BCK seems to be an important and regulated feature for the fueling of membrane-located, ATP-dependent processes, stressing again the importance of local rather than global ATP concentrations.

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubule-associated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

International audience; Resveratrol is attracting much interest because of its potential to decrease body weight and increase life span, influencing liver and muscle function by increasing mitochondrial mass and energy expenditure. Even though resveratrol was already shown to reduce the adipose tissue mass in animal models, its effects on mitochondrial mass and network structure in adipocytes have not yet been studied. For this purpose, we investigated the effect of resveratrol on mitochondrial mass increase and remodeling during adipogenic differentiation of two in vitro models of adipocyte biology, the murine 3T3-L1 cell line and the human SGBS cell strain. We confirm that resveratrol inhibits lipogenesis in differentiating adipocytes, both mouse and human. We further show that this is linked to inhibition of the normally observed mitochondrial mass increase and mitochondrial remodeling. At the molecular level, the anti-lipogenic effect of resveratrol seems to be mediated by a blunted expression increase and an inhibition of acetyl-CoA carboxylase (ACC). This is one of the consequences of an inhibited insulin-induced signaling via Akt, and maintained signaling via AMP-activated protein kinase. The anti-lipogenic effect of resveratrol is further modulated by expression levels of mitochondrial ATAD3, consistent with the emerging role of this protein as an important regulator of mitochondrial biogenesis and lipogenesis. Our data suggest that resveratrol acts on differentiating preadipocytes by inhibiting insulin signaling, mitochondrial biogenesis, and lipogenesis, and that resveratrol-induced reduction of mitochondrial biogenesis and lipid storage contribute to adipose tissue weight loss in animals and humans.

International audience; AMP-activated protein kinase (AMPK) is a central cellular signaling hub that senses and responds to different kinds of stress, mainly those triggered by impaired cellular energy homeostasis. Since this is of major importance for the heart, the kinase plays important roles for cardiovascular function in human health and disease. Here, we review recent progress on the molecular structure and role of AMPK and summarize regulation and biological actions of the AMPK pathway, in particular those relevant for the heart. Activation of the kinase is involved in the myocardial response to ischemia, pressure overload, and heart failure. Pharmacological activation of AMPK may prove to be a useful therapeutic strategy in the treatment of these pathologies. (PDF) AMP-Activated Protein Kinase: A Metabolic Stress Sensor in the Heart. Available from: https://www.researchgate.net/publication/283756966_AMP-Activated_Protein_Kinase_A_Metabolic_Stress_Sensor_in_the_Heart [accessed Dec 10 2018].

International audience; Historically, cellular trafficking of lipids has received much less attention than protein trafficking, mostly because its biological importance was underestimated, involved sorting and translocation mechanisms were not known, and analytical tools were limiting. This has changed during the last decade, and we discuss here some progress made in respect to mitochondria and the trafficking of phospholipids, in particular cardiolipin. Different membrane contact site or junction complexes and putative lipid transfer proteins for intra- and intermembrane lipid translocation have been described, involving mitochondrial inner and outer membrane, and the adjacent membranes of the endoplasmic reticulum. An image emerges how cardiolipin precursors, remodeling intermediates, mature cardiolipin and its oxidation products could migrate between membranes, and how this trafficking is involved in cardiolipin biosynthesis and cell signaling events. Particular emphasis in this review is given to mitochondrial nucleoside diphosphate kinase D and mitochondrial creatine kinases, which emerge to have roles in both, membrane junction formation and lipid transfer.

International audience; Integrated mechanisms of regulation of energy metabolism at cellular, tissue, and organ levels are analyzed from a systems biology perspective. These integrated mechanisms comprise the coordinated function of three cycles of mass and energy transfer and conversion: (1) the Randle cycle of substrate supply, (2) the Krebs cycle coupled with energy transformation in mitochondrial oxidative phosphorylation, and (3) the kinase cycles of intracellular energy transfer and signal transduction for regulation of energy fluxes. These cycles are extended and partially governed by information transfer systems like those linked to protein kinase signaling. In the heart, these cycles are closely related to the Ca2+ cycle during excitation–contraction coupling. According to the view of integrated metabolic cycles, the phosphocreatine/creatine kinase system represents a most important subsystem determining the efficiency of regulation of metabolic and energy fluxes in heart, brain, and oxidative skeletal muscles. It carries about 80 % of the energy flux between mitochondria and cytoplasm in heart. The substrate uptake, respiration rate, and energy fluxes are regulated in response to workload via phosphotransfer pathways and Ca2+ cycling. We propose integrated network mechanisms to explain the linear relationship between myocardial oxygen consumption and heart work output under conditions of metabolic stability (metabolic aspect of Frank–Starling’s law of the heart). The efficiency of energy transfer, force of contraction, and metabolic regulation of respiration and energy fluxes depend upon the intracellular concentration of total creatine, which is decreased in heart failure. The role of creatine, creatine kinase, and adenylate kinase phosphotransfer and AMP-activated protein kinase (AMPK) signaling systems and their interrelationship with substrate supply and Ca2+ cycles are analyzed. Finally, an introduction to the AMPK signaling network is provided with a particular emphasis on the heart in health and disease.

International audience; Nm23-H4/NDPK-D forms symmetrical homohexameric complexes in the mitochondrial intermembrane space. The well established function of the enzyme is phosphotransfer activity as a nucleoside diphosphate kinase (NDPK), using mitochondrial ATP to regenerate nucleoside triphosphates. Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to inner mitochondrial membranes. We show here that such protein/lipid complexes inhibit NDPK activity but are necessary for Nm23-H4 to function in selective intermembrane lipid transfer. Nm23-H4-deficient HeLa cells expressing either wild-type Nm23-H4 or a membrane-binding deficient mutant were analyzed by membrane fractionation and LC-ESI-MS. Data revealed that wild-type Nm24-H4 increased cardiolipin content in the outer mitochondrial membrane as compared to mutant enzyme. This correlated with higher susceptibility of wild-type enzyme expressing cells to rotenone-induced apoptosis as seen by increased annexin V binding, elevated caspase 3/7 activity and stimulated release of cytochrome c into the cytosol. Molecular modeling of Nm23-H4 binding with cardiolipin reveals potential intermembrane transfer mechanisms. We propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function, allowing either for phosphotransfer or for cardiolipin transfer, with a role in apoptotic signaling.

The nucleoside diphosphate kinase Nm23-H4/NDPK-D forms symmetrical hexameric complexes in the mitochondrial intermembrane space with phosphotransfer activity using mitochondrial ATP to regenerate nucleoside triphosphates. We demonstrate the complex formation between Nm23-H4 and mitochondrial GTPase OPA1 in rat liver, suggesting its involvement in local and direct GTP delivery. Similar to OPA1, Nm23-H4 is further known to strongly bind in vitro to anionic phospholipids, mainly cardiolipin, and in vivo to the inner mitochondrial membrane. We show here that such protein-lipid complexes inhibit nucleoside diphosphate kinase activity but are necessary for another function of Nm23-H4, selective intermembrane lipid transfer. Mitochondrial lipid distribution was analyzed by liquid chromatography-mass spectrometry using HeLa cells expressing either wild-type Nm23-H4 or a membrane binding-deficient mutant at a site predicted based on molecular modeling to be crucial for cardiolipin binding and transfer mechanism. We found that wild type, but not the mutant enzyme, selectively increased the content of cardiolipin in the outer mitochondrial membrane, but the distribution of other more abundant phospholipids (e.g. phosphatidylcholine) remained unchanged. HeLa cells expressing the wild-type enzyme showed increased accumulation of Bax in mitochondria and were sensitized to rotenone-induced apoptosis as revealed by stimulated release of cytochrome c into the cytosol, elevated caspase 3/7 activity, and increased annexin V binding. Based on these data and molecular modeling, we propose that Nm23-H4 acts as a lipid-dependent mitochondrial switch with dual function in phosphotransfer serving local GTP supply and cardiolipin transfer for apoptotic signaling and putative other functions.

Background Ubiquitous mitochondrial creatine kinase (uMtCK) accumulates as macroenzyme creatine kinase type 2 (macro CK2) in the serum of HIV-infected patients under a tenofovir disoproxil fumarate (TDF)-containing antiretroviral regimen. The genesis and clinical significance of this finding is unclear. Methods A prospective observational 5-year follow-up study was performed on those patients in which macro CK2 appearance was initially described (‘TDF switch study’ cohort). In addition, tenofovir (TFV), its prodrug TDF and its active, intracellular derivative TFV diphosphate (TDP) were tested in vitro for their effects on different key properties of uMtCK to clarify possible interactions of uMtCK with TFV compounds. Results In just under 5 years of continuous TDF treatment, only 4/12 (33%) patients remained macro CK2-positive, whereas 8/12 (66%) originally positive patients were macro CK2-negative at the end of follow-up. Prospective clinical follow-up data indicate that macro CK2 appearance under TDF is not associated with significant cell damage or occurrence of malignancies. A trend towards grade 1 hypophosphataemia suggests subclinical proximal tubular dysfunction in macro-CK2-positive patients, although it was not associated with a significant decrease in estimated glomerular filtration rate. In vitro, TFV, TDF and TDP did not interfere with uMtCK enzyme activity as competitive inhibitors or pseudo-substrates, but TFV and TDF stabilized the native uMtCK octameric structure in dilute solutions. Conclusions Appearance of octameric uMtCK as macro CK2 in the serum of TDF-treated patients is suggested to result from a combination of low-level mitochondrial damage caused by subclinical renal tubular dysfunction together with possible compensatory uMtCK overexpression and a putative concomitant stabilization of uMtCK octamers by higher levels of TFV in proximal tubules.

International audience; Mitochondrial isoforms of creatine kinase (MtCK) and nucleoside diphosphate kinase (NDPK-D) are not phylogenetically related but share functionally important properties. They both use mitochondrially generated ATP with the ultimate goal of maintaining proper nucleotide pools, are located in the intermembrane/cristae space, have symmetrical oligomeric structures, and show high affinity binding to anionic phospholipids, in particular cardiolipin. The structural basis and functional consequences of the cardiolipin interaction have been studied and are discussed in detail in this review. They mainly result in a functional interaction of MtCK and NDPK-D with inner membrane adenylate translocator, probably by forming proteolipid complexes. These interactions allow for privileged exchange of metabolites (channeling) that ultimately regulate mitochondrial respiration. Further functions of the MtCK/membrane interaction include formation of cardiolipin membrane patches, stabilization of mitochondria and a role in apoptotic signaling, as well as in case of both kinases, a role in facilitating lipid transfer between two membranes. Finally, disturbed cardiolipin interactions of MtCK, NDPK-D and other proteins like cytochrome c and truncated Bid are discussed more generally in the context of apoptosis and necrosis.

International audience; Nucleoside diphosphate kinases (NDPKs/Nm23), responsible for intracellular di- and tri-phosphonucleoside homeostasis, play multi-faceted roles in cellular energetic, signaling, proliferation, differentiation and tumor invasion. The mitochondrial NDPK-D, the NME4 gene product, is a peripheral protein of the inner membrane. Several new aspects of the interaction of NDPK-D with the inner mitochondrial membrane have been recently characterized. Surface plasmon resonance analysis using recombinant NDPK-D and different phospholipid liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin, a phospholipid located mostly in the mitochondrial inner membrane. Mutation of the central arginine (R90) in a surface exposed cationic RRK motif unique to NDPK-D strongly reduced phospholipid interaction in vitro and in vivo. Stable expression of NDPK-D proteins in HeLa cells naturally almost devoid of this isoform revealed a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the enzyme. Owing to its symmetrical hexameric structure exposing membrane binding motifs on two opposite sides, NDPK-D could bridge liposomes containing anionic phospholipids and promote lipid transfer between them. In vivo, NDPK-D could induce intermembrane contacts and facilitate lipid movements between mitochondrial membranes. Most of these properties are reminiscent to those of the mitochondrial creatine kinase. We review here the common properties of both kinases and we discuss their potential roles in mitochondrial functions such as energy production, apoptosis and mitochondrial dynamics.

International audience; We show in this study that mitochondrial creatine kinase promotes segregation and clustering of cardiolipin in mixed membranes, a phenomenon that has been proposed to occur at contact sites in the mitochondria. This property of mitochondrial creatine kinase is dependent on the native octameric structure of the protein and does not occur after heat-denaturation or with the native dimeric form of the protein. Cardiolipin segregation was demonstrated by differential scanning calorimetry using membranes containing cardiolipin and either dipalmitoylphosphatidylethanolamine or 1-palmitoyl-2-oleoylphosphatidylethanolamine. Addition of the ubiquitous form of mitochondrial creatine kinase leads to the formation of a phosphatidylethanolamine-rich domain as a result of the protein binding preferentially to the cardiolipin. Such phase separation does not occur if cardiolipin is replaced with dioleoyl phosphatidylglycerol. Lipid phase separation is observed with other cardiolipin-binding proteins, including cytochrome c and, to a very small extent, with truncated Bid (t-Bid), as well as with the cationic polypeptide poly-L-lysine, but among these proteins the octameric form of mitochondrial creatine kinase is by far the most effective in causing segregation and clustering of cardiolipin. The proteins included in this study are found at mitochondrial contact sites where they are known to associate with cardiolipin. Domains in mitochondria enriched in cardiolipin play an important role in apoptosis and in energy flux processes.

International audience; Intracellular sensors of cellular energy and nutrient status are emerging as key players in the regulation of cell metabolism in health and disease. AMP-activated protein kinase (AMPK) participates in the control of cellular and whole-body energy balance by its exquisite sensitivity to AMP. AMPK is thus able to sense and to react to an increasing AMP:ATP ratio within a complex upstream and downstream signaling network that responds to different energetic and metabolic stresses. The kinase forms heterotrimers with catalytic a-and regulatory b-, and g-subunits, which exist as different isoforms and splice variants. Global and local cellular ATP:ADP and phosphocreatine:creatine ratios are controlled by the reactions of adenylate kinase and creatine kinase, respectively, which are the first safeguards for keeping cellular ATP:ADP and ATP:AMP ratios high for as long aspossible. However, AMPK operates under different metabolic conditions, with different modes of operation and in subsequent time frames. AMP is present at sub-micromolar concentrations in resting cells and increases strongly as a result of the adenylate kinase reaction when ATP is depleted. It is thus an ideal second messenger for reporting cellular energy status to the sensor AMPK. Full activation of AMPK is more complex. In addition to allosteric stimulation by micromolar AMP concentrations, it involves both covalent activation and inhibition viachanges in phosphorylation of the Thr172 residue of the a-subunits by different upstream kinases and phosphatases. Such activation depends on different kinds of energy stress as well as on hormones and other signals, including changes in calcium. As a consequence, AMPK aims not only at cellular energy homeostasis but also at whole-body energy balance. Once activated, AMPK induces compensatory measures to maintain cellular ATP:ADP ratios for cell survival. It regulates a large number of downstream targets, shutting down anabolic pathways and stimulating catabolic pathways, thus simultaneously sparing limited energy resources and acquiring extra energy, respectively. AMPK acts at two control levels that are responsible for acute and chronic responses: first by directly affecting the activity of key enzymes, e.g., in glucose and fat metabolism, and second by longer-term transcriptional control of key players of these metabolic pathways. However, new evidence indicates that AMPK also participates in the control of non-metabolic processes such as cell proliferation and cell cycle. Lastly, the kinase complex has been proposed as a potential drug target in treatment of type II diabetes, because its activation both stimulates insulin-independent glucose uptake in the periphery and suppresses glucose production by the liver. More recently, the discovery of AMPK activation by the tumor suppressor LKB1 suggests a strong connection between metabolic signaling and cancer.

International audience; Doxorubicin (DXR) is a widely used and efficient anticancer drug. However, its application is limited by the risk of severe cardiotoxicity. Impairment of cardiac high-energy phosphate homeostasis is an important manifestation of both acute and chronic DXR cardiotoxic action. Using the Langendorff model of the perfused rat heart, we characterized the acute effects of 1-h perfusion with 2 or 20 microM DXR on two key kinases in cardiac energy metabolism, creatine kinase (CK) and AMP-activated protein kinase (AMPK), and related them to functional responses of the perfused heart and structural integrity of the contractile apparatus as well as drug accumulation in cardiomyocytes. DXR-induced changes in CK were dependent on the isoenzyme, with a shift in protein levels of cytosolic isoenzymes from muscle-type CK to brain-type CK, and a destabilization of octamers of the mitochondrial isoenzyme (sarcometric mitochondrial CK) accompanied by drug accumulation in mitochondria. Interestingly, DXR rapidly reduced the protein level and phosphorylation of AMPK as well as phosphorylation of its target, acetyl-CoA-carboxylase. AMPK was strongly affected already at 2 microM DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate but became significant at 20 microM DXR. Only at 2 microM DXR did upregulation of brain-type CK compensate for inactivation of other isoenzymes. These results suggest that an impairment of kinase systems regulating cellular energy homeostasis is involved in the development of DXR cardiotoxicity.

International audience; High affinity interaction between octameric mitochondrial creatine kinase (MtCK) and the phospholipid cardiolipin in the inner mitochondrial membrane plays an important role in metabolite channeling between MtCK and inner membrane adenylate translocator, which itself is tightly bound to cardiolipin. Three C-terminal basic residues revealed as putative cardiolipin anchors in the x-ray structures of MtCK and corresponding to lysines in human sarcomeric MtCK (sMtCK) were exchanged by in vitro mutagenesis (K369A/E, K379Q/A/E, K380Q/A/E) to yield double and triple mutants. sMtCK proteins were bacterially expressed, purified to homogeneity, and verified for structural integrity by enzymatic activity, gel filtration chromatography, and CD spectroscopy. Interaction with cardiolipin and other acidic phospholipids was quantitatively analyzed by light scattering, surface plasmon resonance, and fluorescence spectroscopy. All mutant sMtCKs showed a strong decrease in vesicle cross-linking, membrane affinity, binding capacity, membrane ordering capability, and binding-induced changes in protein structure as compared with wild type. These effects did not depend on the nature of the replacing amino acid but on the number of exchanged lysines. They were moderate for Lys-379/Lys-380 double mutants but pronounced for triple mutants, with a 30-fold lower membrane affinity and an entire lack of alterations in protein structure compared with wild-type sMtCK. However, even triple mutants partially maintained an increased order of cardiolipin-containing membranes. Thus, the three C-terminal lysines determine high affinity sMtCK/cardiolipin interaction and its effects on MtCK structure, whereas low level binding and some effect on membrane fluidity depend on other structural components. These results are discussed in regard to MtCK microcompartments and evolution.

International audience; Anthracyclines are among the most efficient drugs of cancer chemotherapy, but their use is limited by a significant risk of cardiotoxicity, which is still far from being understood. This study investigates whether impairment of mitochondrial creatine kinase (MtCK), a key enzyme in cellular energy metabolism, could be involved in anthracycline cardiotoxicity. We have analyzed the effects of three anthracyclines, doxorubicin, daunorubicin, and idarubicin, on two MtCK isoenzymes, sarcomeric/cardiac sMtCK and ubiquitous uMtCK, from human and chicken. Using surface plasmon resonance, gel filtration, and enzyme assays, we have quantified properties that are of basic importance for MtCK functioning in vivo: membrane binding, octameric state, and enzymatic activity. Anthracyclines significantly impaired all three properties with differences in dose-, time-, and drug-dependence. Membrane binding and enzymatic activity were already affected at low anthracycline concentrations (5-100 microM), indicating high clinical relevance. Effects on membrane binding were immediate, probably because of competitive binding of the drug to cardiolipin. In contrast, dissociation of MtCK octamers into dimers, enzymatic inactivation and cross-linking occurred only after hours to days. Different protection assays suggest that the deleterious effects were caused by oxidative damage, mainly affecting the highly susceptible MtCK cysteines, followed by generation of free oxygen radicals at higher drug concentrations. Enzymatic inactivation occurred mainly at the active site and involved Cys278, as indicated by experiments with protective agents and sMtCK mutant C278G. All anthracycline effects were significantly more pronounced for sMtCK than for uMtCK. These in vitro results suggest that sMtCK damage may play a role in anthracycline cardiotoxicity.

Epitopes differing among isoenzymes of creatine kinase (CK) are apparently limited in number and poorly immunogenic in vivo. Especially for the BB-CK isoenzyme, very few monoclonal antibodies (mAb) are available. Here, we use in vitro selection with a synthetic human phage display antibody library and develop isoenzyme competition and peptide panning strategies to obtain human single chain Fv (scFv) antibodies against specific CK isoenzymes. We isolated and characterized seven scFv clones that recognize native as well as denatured cytosolic BB-CK in ELISA, immunoblot, immunofluorescence histochemistry and surface plasmon resonance (SPR) spectroscopy. To a variable but minor degree, they also react with cytosolic MM-CK, but not with mitochondrial CK isoenzymes. Epitope mapping revealed that the scFv antibodies recognize different BB-CK epitopes, including the N-terminus and the isoenzyme-specific box, a highly conserved sequence of unknown function for which no mAb were available so far. With a KD of 3.5–9.610 7 M, the isolated scFv compare favorably with mouse mAb and may overcome certain of their limitations. Our results demonstrate the advantages of in vitro antibody selection for the generation of isoenzyme-specific antibodies. D 2002 Elsevier Science B.V. All rights reserved.

International audience; Mitochondrial creatine kinase (MtCK) co-localizes with mitochondrial porin (voltage-dependent anion channel) and adenine nucleotide translocator in mitochondrial contact sites. A specific, direct protein-protein interaction between MtCK and mitochondrial porin was demonstrated using surface plasmon resonance spectroscopy. This interaction was independent of the immobilized binding partner (porin reconstituted in liposomes or MtCK) or the analyzed isoform (chicken sarcomeric MtCK or human ubiquitous MtCK, human recombinant porin, or purified bovine porin). Increased ionic strength reduced the binding of MtCK to porin, suggesting predominantly ionic interactions. By contrast, micromolar concentrations of Ca(2+) increased the amount of bound MtCK, indicating a physiological regulation of complex formation. No interaction of MtCK with reconstituted adenine nucleotide translocator was detectable in our experimental setup. The relevance of these findings for structure and function of mitochondrial contact sites is discussed.

Doxorubicin (DXR) is an efficient anticancer drug, the use of which is limited by seriouscardiotoxicity. Despite decades of research, its pathogenic mechanisms are not fully understood, andefficient preventive or curative strategies are not available. Here we address the question whethermechanisms in cardiac cell signaling contribute to the toxicity phenotype. Using experimental modelsfor acute (DXR-perfused, isolated rat hearts) or chronic toxicity (rats injected with DXR), we conducteda targeted study (focusing on energy signaling pathways) and two non-biased studies(phosphoproteomics and transcriptomics). The combined data reveal DXR-induced alterations inphosphorylation (activation) status or gene expression of proteins in mainly three functional domains:energy metabolism, stress responses, and sarcomere structure. (i) Energy metabolism: We confirm aparadox inhibition of AMPK signaling, that is rather due to inhibitory cross-talk with related signalingpartners (Akt, ERK) than impaired AMPK upstream signaling. We also show, among others, theincrease of inhibitory phosphorylation of pyruvate dehydrogenase, slowing down Krebs cycle, but alsoa compensating upregulation of glycolysis and Krebs cycle enzyme transcripts. (ii) Stress-responses:In our models, DXR generates energetic, oxidative and genotoxic stress, but only some compensatorystress responses are activated (DNA-PK–Akt–GSK3 pathway, chaperones). Many others seem to beinhibited, suggesting a blunted response to stress as component of DXR toxicity. (iii) Sarcomerestructure/function: We detect increased phosphorylation of desmin and reduced transcripts essentialfor e.g. normal heart development as potential causes for a disorganized myofibrillar network. Inconclusion, these results reveal some novel potential mechanisms of DXR-induced cardiotoxicity andsuggest new targets for protective strategies., La doxorubicine (DXR) est l’un des médicaments les plus efficaces en chimiothérapie, mais sonapplication clinique est limitée par ses effets cardiotoxiques. Malgré des décennies de recherche, sesmécanismes pathogéniques ne sont toujours pas entièrement compris. Il s’ensuit qu’aucun traitementsatisfaisant, curatif ou préventif, n’existe. Dans cette étude, nous recherchons les mécanismes designalisation cellulaire impliqués. Deux modèles expérimentaux de toxicités, aigue d’une part (coeurisolé et perfusé de rat avec la DXR), et chronique d’autre part (rat traité à la DXR), ont permis deréaliser une étude ciblée (sur les voies de signalisation énergétiques) et deux études systémiques(phosphoprotéomique et transcriptomique). Les résultats combinés de ces travaux ont montré que laDXR modifiait le niveau de phosphorylation (activation) ou l’expression génique de protéinesimpliquées dans trois domaines fonctionnels distincts : métabolisme énergétique, réponses au stress,et structure/fonction du sarcomère. (i) Métabolisme énergétique : nous avons confirmé la surprenanteinhibition de l’AMPK, probablement provoquée par un contrôle négatif exercé par des partenaires designalisation (Akt et ERK), plutôt que par une modification des kinases activatrices en amont. Nousavons également montré l’augmentation du niveau de phosphorylation de la PDH, ce qui, en inhibantl’enzyme, ralentit le cycle de Krebs. Cependant, nous avons également observé un phénomènecompensatoire de surexpression de gènes codant pour des enzymes de la glycolyse et du cycle deKrebs ; (ii) Réponses au stress : dans nos modèles, la DXR génère des stress énergétique,génotoxique et oxydatif. Cependant, seuls quelques mécanismes compensatoires sont activés (lesvoies de signalisation de DNA-PK–Akt–GSK3, diverses chaperonnes). Les autres semblent êtreinhibées suggérant que l’amoindricement des réponses au stress serait un des mécanismes de lacardiotoxicité de la DXR; (iii) Structure/fonction du sarcomère: L’augmentation de la phosphorylationde la desmine ainsi que la réduction du nombre de transcrits codant pour des protéines essentiellesau développement cardiaque normal pourraient être la cause de la désorganisation du réseaumyofibrillaire. En conclusion, ces résultats révèlent potentiellement de nouveaux mécanismes de lacardiotoxicité induite par la DXR et permettent d’envisager de nouvelles cibles moléculaires pour ledéveloppement de stratégies protectrices.