Laurence Kay

Permeabilized cell and skinned fiber techniques in studies of mitochondrial function in vivo

In this chapter we describe in details the permeabilized cell and skinned fiber techniques and their applications for studies of mitochondrial function in vivo. The experience of more than 10 years of research in four countries is summarized. The use of saponin in very low concentration (50–100 μg/ml) for permeabilisation of the sarcolemma leaves all intracellular structures, including mitochondria, completely intact. The intactness of mitochondrial function in these skinned muscle fibers is demonstrated in this work by multiple methods, such as NADH and flavoprotein fluorescence studies, fluorescence imaging, confocal immunofluorescence microscopy and respiratory analysis. Permeabilized cell and skinned fiber techniques have several very significant advantages for studies of mitochondrial function, in comparison with the traditional methods of use of isolated mitochondria: (1) very small tissue samples are required; (2) all cellular population of mitochondria can be investigated; (3) most important, however, is that mitochondria are studied in their natural surrounding. The results of research by using this method show the existence of several new phenomenon — tissue dependence of the mechanism of regulation of mitochondrial respiration, and activation of respiration by selective proteolysis. These phenomena are explained by interaction of mitochondria with other cellular structures in vivo. The details of experimental studies with use of these techniques and problems of kinetic analysis of the results are discussed. Examples of large-scale clinical application of these methods are given. (Mol Cell Biochem 184: 81–100, 1998)

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.

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

AIMSnCardiotoxic 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.nnnMETHODS AND RESULTSnEffects 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.nnnCONCLUSIONnOur 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.

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.

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.

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 Ca2+ 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.

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.

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

BACKGROUNDnUbiquitous 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.nnnMETHODSnA 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.nnnRESULTSnIn 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.nnnCONCLUSIONSnAppearance 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.

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.