The pleiotropic effects of creatine (Cr) are based mostly on the functions of the enzyme creatine kinase (CK) and its high-energy product phosphocreatine (PCr). Multidisciplinary studies have established molecular, cellular, organ and somatic functions of the CK/PCr system, in particular for cells and tissues with high and intermittent energy fluctuations. These studies include tissue-specific expression and subcellular localization of CK isoforms, high-resolution molecular structures and structure–function relationships, transgenic CK abrogation and reverse genetic approaches. Three energy-related physiological principles emerge, namely that the CK/PCr systems functions as (a) an immediately available temporal energy buffer, (b) a spatial energy buffer or intracellular energy transport system (the CK/PCr energy shuttle or circuit) and (c) a metabolic regulator. The CK/PCr energy shuttle connects sites of ATP production (glycolysis and mitochondrial oxidative phosphorylation) with subcellular sites of ATP utilization (ATPases). Thus, diffusion limitations of ADP and ATP are overcome by PCr/Cr shuttling, as most clearly seen in polar cells such as spermatozoa, retina photoreceptor cells and sensory hair bundles of the inner ear. The CK/PCr system relies on the close exchange of substrates and products between CK isoforms and ATP-generating or -consuming processes. Mitochondrial CK in the mitochondrial outer compartment, for example, is tightly coupled to ATP export via adenine nucleotide transporter or carrier (ANT) and thus ATP-synthesis and respiratory chain activity, releasing PCr into the cytosol. This coupling also reduces formation of reactive oxygen species (ROS) and inhibits mitochondrial permeability transition, an early event in apoptosis. Cr itself may also act as a direct and/or indirect anti-oxidant, while PCr can interact with and protect cellular membranes. Collectively, these factors may well explain the beneficial effects of Cr supplementation. The stimulating effects of Cr for muscle and bone growth and maintenance, and especially in neuroprotection, are now recognized and the first clinical studies are underway. Novel socio-economically relevant applications of Cr supplementation are emerging, e.g. for senior people, intensive care units and dialysis patients, who are notoriously Cr-depleted. Also, Cr will likely be beneficial for the healthy development of premature infants, who after separation from the placenta depend on external Cr. Cr supplementation of pregnant and lactating women, as well as of babies and infants are likely to be of benefit for child development. Last but not least, Cr harbours a global ecological potential as an additive for animal feed, replacing meat- and fish meal for animal (poultry and swine) and fish aqua farming. This may help to alleviate human starvation and at the same time prevent over-fishing of oceans.

Mitochondrial creatine kinase (MtCK), together with cytosolic creatine kinase isoenzymes and the highly diffusible CK reaction product, phosphocreatine, provide a temporal and spatial energy buffer to maintain cellular energy homeostasis. Mitochondrial proteolipid complexes containing MtCK form microcompartments that are involved in channeling energy in form of phosphocreatine rather than ATP into the cytosol. Under situations of compromised cellular energy state, which are often linked to ischemia, oxidative stress and calcium overload, two characteristics of mitochondrial creatine kinase are particularly relevant: its exquisite susceptibility to oxidative modifications and the compensatory up-regulation of its gene expression, in some cases leading to accumulation of crystalline MtCK inclusion bodies in mitochondria that are the clinical hallmarks for mitochondrial cytopathies. Both of these events may either impair or reinforce, respectively, the functions of mitochondrial MtCK complexes in cellular energy supply and protection of mitochondria form the so-called permeability transition leading to apoptosis or necrosis.

Development of cardiac hypertrophy and progression to heart failure entails profound changes in myocardial metabolism, characterized by a switch from fatty acid utilization to glycolysis and lipid accumulation. We report that hypoxia-inducible factor (HIF)1alpha and PPARgamma, key mediators of glycolysis and lipid anabolism, respectively, are jointly upregulated in hypertrophic cardiomyopathy and cooperate to mediate key changes in cardiac metabolism. In response to pathologic stress, HIF1alpha activates glycolytic genes and PPARgamma, whose product, in turn, activates fatty acid uptake and glycerolipid biosynthesis genes. These changes result in increased glycolytic flux and glucose-to-lipid conversion via the glycerol-3-phosphate pathway, apoptosis, and contractile dysfunction. Ventricular deletion of Hif1alpha in mice prevents hypertrophy-induced PPARgamma activation, the consequent metabolic reprogramming, and contractile dysfunction. We propose a model in which activation of the HIF1alpha-PPARgamma axis by pathologic stress underlies key changes in cell metabolism that are characteristic of and contribute to common forms of heart disease.

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. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol 289: H37-H47, 2005. First published March 11, 2005; doi:10.1152/ajpheart.01057.2004 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 M 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 mitochondiral 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 M DXR, even before substantial cardiac dysfunction occurred. Impairment of CK isoenzymes was mostly moderate but became significant at 20 M DXR. Only at 2 M 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. creatine kinase; adenosine 5Ј-monophosphate-activated protein kinase; anthracyclines; cardiac energetics; cardiotoxicity DOXORUBICIN (DXR) is a widely used and very efficient anticancer drug. However, its administration is limited by the risk of severe cardiotoxicity (31, 41). An important manifestation of DXR cardiotoxicity is an impaired cardiac high-energy phosphate metabolism. Acute and chronic consequences of DXR administration include compromised mitochondrial functions, such as respiration and generation of high-energy phosphates (30) and lowered phosphocreatine-to-creatine (PCr/Cr), PCr-to-ATP (PCr/ATP), and ATP-to-ADP (ATP/ADP) ratios as well as compromised calcium homeostasis (8,29,31). However, the involved molecular mechanisms are not yet entirely understood.The maintenance of stable concentrations of myocardial high-energy phosphates, ATP, and PCr is a fundamental principle in the vertebrate heart. The PCr/ATP ratio is constant across species, as well as within a species, over a wide range of physiological cardiac workloads (12). Different regulatory systems have evolved to control cellular energy production and utilization. A key player of this regulatory network is the concerted action of several kinase systems, in particular, the energy transfer and buffer system of c...

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 microtubuleassociated 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.

Nucleoside diphosphate kinases (NDPK), 3 encoded by NME genes (also called NM23), catalyze the exchange of ␥-phosphate between di-and triphosphonucleosides and participate in the regulation of intracellular nucleotide homeostasis. They mainly utilize ATP formed by oxidative phosphorylation to synthesize the other triphosphonucleosides, in particular GTP (1). Given the poor substrate selectivity of NDPKs, it is assumed that specificity could arise from the presence of different isoforms at different subcellular localizations. Associated in networks with other nucleotide-metabolizing enzymes such as adenylate kinases, creatine kinases, and glycolytic enzymes, NDPKs participate in high energy phosphoryl transfer and signal communication in the cell (2). Up to now nine genes encoding NDPK or NDPK-like proteins have been identified (3, 4), but little is known about their respective role within the cell. The most studied, NDPK-A and -B, encoded by NME1 and NME2 genes, respectively, play a key role in tumor progression and metastasis dissemination (5, 6).NDPK activity has been found associated with different cellular compartments, such as cytosol, nucleus, plasma membrane, and mitochondria. Precise localization in the latter organelles has been a matter of debate. Depending on species and tissue examined, NDPK activity was reported in both the matrix and the intermembrane/cristae space (7), including the so-called contact sites between inner and outer membrane (8 -10). In mammalian liver (rat and rabbit), the NDPK activity was mainly associated with an extra-matrix compartment, probably the intermembrane/cristae space, whereas in heart activity was more abundant in the matrix (11). For mitochondrial NDPK in matrix, many functions have been proposed ranging from nucleotide supply for mitochondrial nucleic acid and protein synthesis to functional interaction with the Krebs * This work was supported by the Germaine de Stael Program for FrancoSwiss collaboration (to U. S. and M.-L. L.), the Agence Nationale de la Recherche (Chaire d'Excellence (to U. S.)), the Marie Curie Intraeuropean Fellowship of the European Community (to M. T.-S.), INSERM, and grants from the Groupement des Entreprises Françaises contre le Cancer and from the Association pour la Recherche contre le Cancer (to M.-L. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Background: Nm23-H4 is a mitochondrial nucleoside diphosphate kinase that binds mitochondrial membranes. Results: Nm23-H4 interaction with GTPase OPA1 provides a local GTP supply; its interaction with anionic phospholipids inhibits kinase activity but allows intermembrane cardiolipin transfer and sensitizes for apoptosis. Conclusion: Nm23-H4 is a bifunctional switch operated by cardiolipin. Significance: The cardiolipin transfer property has various implications, e.g. for lipid metabolism and apoptosis.