Multi-Method Quantification of Acetyl-Coenzyme A and Further Acyl-Coenzyme A Species in Normal and Ischemic Rat Liver.

Thioesters of coenzyme A (CoA) carrying different acyl chains (acyl-CoAs) are central intermediates of many metabolic pathways and donor molecules for protein lysine acylation. Acyl-CoA species largely differ in terms of cellular concentrations and physico-chemical properties, rendering their analysis challenging. Here, we compare several approaches to quantify cellular acyl-CoA concentrations in normal and ischemic rat liver, using HPLC and LC-MS/MS for multi-acyl-CoA analysis, as well as NMR, fluorimetric and spectrophotometric techniques for the quantification of acetyl-CoAs. In particular, we describe a simple LC-MS/MS protocol that is suitable for the relative quantification of short and medium-chain acyl-CoA species. We show that ischemia induces specific changes in the short-chain acyl-CoA relative concentrations, while mild ischemia (1-2 min), although reducing succinyl-CoA, has little effects on acetyl-CoA, and even increases some acyl-CoA species upstream of the tricarboxylic acid cycle. In contrast, advanced ischemia (5-6 min) also reduces acetyl-CoA levels. Our approach provides the keys to accessing the acyl-CoA metabolome for a more in-depth analysis of metabolism, protein acylation and epigenetics.

Supplementing Soy-Based Diet with Creatine in Rats: Implications for Cardiac Cell Signaling and Response to Doxorubicin.

Nutritional habits can have a significant impact on cardiovascular health and disease. This may also apply to cardiotoxicity caused as a frequent side effect of chemotherapeutic drugs, such as doxorubicin (DXR). The aim of this work was to analyze if diet, in particular creatine (Cr) supplementation, can modulate cardiac biochemical (energy status, oxidative damage and antioxidant capacity, DNA integrity, cell signaling) and functional parameters at baseline and upon DXR treatment. Here, male Wistar rats were fed for 4 weeks with either standard rodent diet (NORMAL), soy-based diet (SOY), or Cr-supplemented soy-based diet (SOY + Cr). Hearts were either freeze-clamped in situ or following ex vivo Langendorff perfusion without or with 25 µM DXR and after recording cardiac function. The diets had distinct cardiac effects. Soy-based diet (SOY vs. NORMAL) did not alter cardiac performance but increased phosphorylation of acetyl-CoA carboxylase (ACC), indicating activation of rather pro-catabolic AMP-activated protein kinase (AMPK) signaling, consistent with increased ADP/ATP ratios and lower lipid peroxidation. Creatine addition to the soy-based diet (SOY + Cr vs. SOY) slightly increased left ventricular developed pressure (LVDP) and contractility dp/dt, as measured at baseline in perfused heart, and resulted in activation of the rather pro-anabolic protein kinases Akt and ERK. Challenging perfused heart with DXR, as analyzed across all nutritional regimens, deteriorated most cardiac functional parameters and also altered activation of the AMPK, ERK, and Akt signaling pathways. Despite partial reprogramming of cell signaling and metabolism in the rat heart, diet did not modify the functional response to supraclinical DXR concentrations in the used acute cardiotoxicity model. However, the long-term effect of these diets on cardiac sensitivity to chronic and clinically relevant DXR doses remains to be established.

Role of Cardiac AMP-Activated Protein Kinase in a Non-pathological Setting: Evidence From Cardiomyocyte-Specific, Inducible AMP-Activated Protein Kinase α1α2-Knockout Mice.

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 Proteolipid Complexes of Creatine Kinase.

Isoforms of creatine kinase (CK) generate and use phosphocreatine, a concentrated and highly diffusible cellular "high energy" intermediate, for the main purpose of energy buffering and transfer in order to maintain cellular energy homeostasis. The mitochondrial CK isoform (mtCK) localizes to the mitochondrial intermembrane and cristae space, where it assembles into peripherally membrane-bound, large cuboidal homooctamers. These are part of proteolipid complexes wherein mtCK directly interacts with cardiolipin and other anionic phospholipids, as well as with the VDAC channel in the outer membrane. This leads to a stabilization and cross-linking of inner and outer mitochondrial membrane, forming so-called contact sites. Also the adenine nucleotide translocator of the inner membrane can be recruited into these proteolipid complexes, probably mediated by cardiolipin. The complexes have functions mainly in energy transfer to the cytosol and stimulation of oxidative phosphorylation, but also in restraining formation of reactive oxygen species and apoptosis. In vitro evidence indicates a putative role of mtCK in mitochondrial phospholipid distribution, and most recently a role in thermogenesis has been proposed. This review summarizes the essential structural and functional data of these mtCK complexes and describes in more detail the more recent advances in phospholipid interaction, thermogenesis, cancer and evolution of mtCK.

Creatine kinase in human erythrocytes: A genetic anomaly reveals presence of soluble brain-type isoform.

For maintaining energy homeostasis, creatine kinase (CK) is present at elevated levels in tissues with high and/or fluctuating energy requirements such as muscle, brain, and epithelia, while there is very few CK, if any, in peripheral blood cells. However, an ectopic expression of brain-type creatine kinase (BCK) has been reported for platelets and leukocytes in an autosomal dominant inherited anomaly named CKBE. Here we investigated CK in erythrocytes of CKBE individuals from eight unrelated families. The data revealed a varying but significant increase of CK activity in CKBE individuals as compared to controls, reaching an almost 800-fold increase in two CKBE individuals which also had increased erythrocyte creatine. Immunoblotting with highly specific antibodies confirmed that the expressed CK isoform is BCK. Cell fractionation evidenced soluble BCK, suggesting cytosolic and not membrane localization of erythrocyte CK as reported earlier. These results are discussed in the context of putative CK energy buffering and transfer functions in red blood cells.

Cellular compartmentation of energy metabolism: creatine kinase microcompartments and recruitment of B-type creatine kinase to specific subcellular sites.

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.

Macro CK2 accumulation in tenofovir-treated HIV patients is facilitated by CK oligomer stabilization but is not predictive for pathology.

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.

Inhibition of AMPK signalling by doxorubicin: at the crossroads of the cardiac responses to energetic, oxidative, and genotoxic stress.

AIMS: Cardiotoxic side effects of anthracyclines, the most widely used anticancer drugs, are well documented, while mechanisms involved are not fully elucidated. The cellular energy sensor and regulator AMP-activated protein kinase (AMPK) was suggested as a putative mediator of cardiotoxicity of doxorubicin, the leading anthracycline drug, by our earlier work. Here, we study the interference of doxorubicin with AMPK signalling and potentially involved mechanisms. METHODS AND RESULTS: Effects of doxorubicin on cell signalling are studied in isolated Langendorff-perfused Wistar rat hearts and in hearts from doxorubicin-treated Wistar rats. In both models, doxorubicin induces energetic, oxidative, and genotoxic stress. Despite energy depletion and unaffected AMPK upstream signalling, doxorubicin does not activate the AMPK pathway and even reduces basal phosphorylation of AMPK and its downstream target acetyl-CoA carboxylase. In contrast, oxidative and genotoxic stress do activate pro-survival mitogen-activated protein kinase (MAPK) and Akt pathways, the latter via DNA-dependent protein kinase activation triggered by DNA damage. Combined inhibition of AMPK and activation of Akt and MAPK lead to activation of growth-stimulating mammalian target of rapamycin (mTOR) signalling. CONCLUSION: Our results suggest that in the doxorubicin-challenged heart, a combined energetic, oxidative, and genotoxic stress elicits a specific, hierarchical response where AMPK is inhibited at least partially by the known negative cross-talk with Akt and MAPK pathways, largely triggered by DNA damage signalling. Although such signalling can be protective, e.g. by limiting apoptosis, it primarily induces a negative feedback that increases cellular energy deficits, and via activation of mTOR signalling, it also contributes to the pathological cardiac phenotype in chronic doxorubicin toxicity.

Cardiac phosphoproteome reveals cell signaling events involved in doxorubicin cardiotoxicity.

The successful use of anthracyclines like doxorubicin in chemotherapy is limited by their severe cardiotoxicity. Despite decades of clinical application, a satisfying description of the molecular mechanisms involved and a preventive treatment have not yet been achieved. Here we address doxorubicin-induced changes in cell signaling as a novel potential mediator of doxorubicin toxicity by applying a non-biased screen of the cardiac phosphoproteome. Two-dimensional gel electrophoresis, phosphospecific staining, quantitative image analysis, and MALDI-TOF/TOF mass spectrometry were combined to identify (de)phosphorylation events occurring in the isolated rat heart upon Langendorff-perfusion with clinically relevant (5 µM) and supraclinical concentrations (25 µM) of doxorubicin. This approach identified 22 proteins with a significantly changed phosphorylation status and these results were validated by immunoblotting for selected phosphosites. Overrepresentation of mitochondrial proteins (>40%) identified this compartment as a prime target of doxorubicin. Identified proteins were mainly involved in energy metabolism (e.g. pyruvate dehydrogenase and acyl-CoA dehydrogenase), sarcomere structure and function (e.g. desmin) or chaperone-like activities (e.g. α-crystallin B chain and prohibitin). Changes in phosphorylation of pyruvate dehydrogenase, regulating pyruvate entry into the Krebs cycle, and desmin, maintaining myofibrillar array, are relevant for main symptoms of cardiac dysfunction related to doxorubicin treatment, namely energy imbalance and myofibrillar disorganization. This article is part of a Special Issue entitled: Translational Proteomics.

Early effects of doxorubicin in perfused heart: transcriptional profiling reveals inhibition of cellular stress response genes.

Doxorubicin (DXR) belongs to the most efficient anticancer drugs. However, its clinical application is limited by the risk of severe cardiac-specific toxicity, for which an efficient treatment is missing. Underlying molecular mechanisms are not sufficiently understood so far, but nonbiased, systemic approaches can yield new clues to develop targeted therapies. Here, we applied a genome-wide transcriptome analysis to determine the early cardiac response to DXR in a model characterized earlier, that is, rat heart perfusion with 2 muM DXR, leading to only mild cardiac dysfunction. Single-gene and gene set enrichment analysis of DNA microarrays yielded robust data on cardiac transcriptional reprogramming, including novel DXR-responsive pathways. Main characteristics of transcriptional reprogramming were 1) selective upregulation of individual genes or gene sets together with widespread downregulation of gene expression; 2) repression of numerous transcripts involved in cardiac stress response and stress signaling; 3) modulation of genes with cardiac remodeling capacity; 4) upregulation of "energy-related" pathways; and 5) similarities to the transcriptional response of cancer cells. Some early responses like the induction of glycolytic and Krebs cycle genes may have compensatory function. Only minor changes in the cardiac energy status or the respiratory activity of permeabilized cardiac fibers have been observed. Other responses potentially contribute to acute and also chronic toxicity, in particular, those in stress-responsive and cardiac remodeling transcripts. We propose that a blunted response to stress and reduced "danger signaling" is a prime component of toxic DXR action and can drive cardiac cells into pathology.

Mitochondrial kinases and their molecular interaction with cardiolipin.

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.

Mitochondrial regular arrangement in muscle cells: a "crystal-like" pattern.

The aim of this work was to characterize quantitatively the arrangement of mitochondria in heart and skeletal muscles. We studied confocal images of mitochondria in nonfixed cardiomyocytes and fibers from soleus and white gastrocnemius muscles of adult rats. The arrangement of intermyofibrillar mitochondria was analyzed by estimating the densities of distribution of mitochondrial centers relative to each other (probability density function). In cardiomyocytes (1,820 mitochondrial centers marked), neighboring mitochondria are aligned along a rectangle, with distance between the centers equal to 1.97 +/- 0.43 and 1.43 +/- 0.43 microm in the longitudinal and transverse directions, respectively. In soleus (1,659 mitochondrial centers marked) and white gastrocnemius (621 pairs of mitochondria marked), mitochondria are mainly organized in pairs at the I-band level. Because of this organization, there are two distances characterizing mitochondrial distribution in the longitudinal direction in these muscles. The distance between mitochondrial centers in the longitudinal direction within the same I band is 0.91 +/- 0.11 and 0.61 +/- 0.07 microm in soleus and white gastrocnemius, respectively. The distance between mitochondrial centers in different I bands is approximately 3.7 and approximately 3.3 microm in soleus and gastrocnemius, respectively. In the transverse direction, the mitochondria are packed considerably closer to each other in soleus than in white gastrocnemius, with the distance equal to 0.75 +/- 0.22 microm in soleus and 1.09 +/- 0.41 microm in gastrocnemius. Our results show that intermyofibrillar mitochondria are arranged in a highly ordered crystal-like pattern in a muscle-specific manner with relatively small deviation in the distances between neighboring mitochondria. This is consistent with the concept of the unitary nature of the organization of the muscle energy metabolism.

Possible role of cytoskeleton in intracellular arrangement and regulation of mitochondria.

The origin of significant differences between the apparent affinities of heart mitochondrial respiration for exogenous ADP in isolated mitochondria in vitro and in permeabilized cardiomyocytes or skinned fibres in situ is critically analysed. All experimental data demonstrate the importance of structural factors of intracellular arrangement of mitochondria into functional complexes with myofibrils and sarcoplasmic reticulum in oxidative muscle cells and the control of outer mitochondrial membrane permeability. It has been shown that the high apparent K(m) for exogenous ADP (250-350 mM) in permeabilized cells and in ghost cells (without myosin) and fibres (diameter 15-20 mm) is independent of intrinsic MgATPase activity. However, the K(m) may be decreased significantly by a selective proteolytic treatment, which also destroys the regular arrangement of mitochondria between sarcomeres and increases the accessibility of endogenous ADP to the exogenous pyruvate kinase-phosphoenolpyruvate system. The confocal microscopy was used to study the changes in intracellular distribution of mitochondria and localization of cytoskeletal proteins, such as desmin, tubulin and plectin in permeabilized cardiac cells during short proteolytic treatment. The results show the rapid collapse of microtubular and plectin networks but not of desmin localization under these conditions. These results point to the participation of cytoskeletal proteins in the intracellular organization and control of mitochondrial function in the cells in vivo, where mitochondria are incorporated into functional complexes with sarcomeres and sarcoplasmic reticulum.

An increase in the myocardial PCr/ATP ratio in GLUT4 null mice.

ATP and creatine phosphate (PCr) are prime myocardial high-energy phosphates. Their relative concentrations are conserved among mammalian species and across a range of physiologic cardiac workloads. The cardiac PCr/ATP ratio is decreased with several pathologic conditions, such as ischemia and heart failure, but there are no reports of an increase in the cardiac PCr/ATP ratio in any species or with interventions. We studied the in vivo energetics in transgenic mice lacking expression of the glucose transport protein GLUT4 (G4N) and observed a significant 60% increase in the myocardial PCr/ATP ratio in G4N that was confirmed in three different experimental settings including intact animals. The higher PCr/ATP in G4N is cardiac-specific and is due to higher total cardiac creatine (CR) concentrations in G4N than in wild-type (WT). However, [ATP], [ADP], and -DG(-ATP) did not differ between the strains. Expression of the creatine transport protein (CreaT) that is responsible for creatine uptake in myocytes was preserved in G4N cardiac tissue. These observations demonstrate, for the first time to our knowledge, that G4N manifest a unique increase in the cardiac PCr/ATP ratio, which suggests a novel genetic strategy for increasing myocardial creatine levels.

Creatine supplementation reduces skeletal muscle degeneration and enhances mitochondrial function in mdx mice.

The mdx mouse serves as animal model for Duchenne muscular dystrophy. Energy status in muscles of mdx mice is impaired and we have demonstrated recently that the energy precursor creatine exerts beneficial effects on mdx skeletal muscle cells in culture. Here we show that feeding a creatine-enriched diet to new-born mdx mice strongly reduced the first wave of muscle necrosis four weeks after birth. Necrosis of the fast-twitch muscle extensor digitorum longus was inhibited by 63+/-14% (P<0.0001) while necrosis of the slow-twitch soleus muscle was not significantly decreased. In addition, using chemically skinned muscle fibres, we found that mitochondrial respiration capacity was decreased by about 25% in mdx-derived fibres and that long-term creatine-feeding restored respiration to wild-type levels. These results provide evidence that creatine supplementation in mdx mice improves muscle health and may provide a scientific basis for its use as adjuvant therapy in Duchenne muscular dystrophy.