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The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.

The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium., Graphical abstract

The identification of novel physiological regulators that stimulate energy expenditure through brown adipose tissue (BAT) activity in substrate catalysis is of utmost importance to understand and treat metabolic diseases. Myoglobin (MB), known to store or transport oxygen in heart and skeletal muscles, has recently been found to bind fatty acids with physiological constants in its oxygenated form (i.e., MBO2). Here, we investigated the in vivo effect of MB expression on BAT activity. In particular, we studied mitochondrial function and lipid metabolism as essential determinants of energy expenditure in this tissue. We show in a MB-null (MBko) mouse model that MB expression in BAT impacts on the activity of brown adipocytes in a twofold manner: i) by elevating mitochondrial density plus maximal respiration capacity, and through that, by stimulating BAT oxidative metabolism along with the organelles` uncoupled respiration; and ii) by influencing the free fatty acids pool towards a palmitate-enriched composition and shifting the lipid droplet (LD) equilibrium towards higher counts of smaller droplets. These metabolic changes were accompanied by the up-regulated expression of thermogenesis markers UCP1, CIDEA, CIDEC, PGC1-α and PPAR-α in the BAT of MB wildtype (MBwt) mice. Along with the emergence of the “browning” BAT morphology, MBwt mice exhibited a leaner phenotype when compared to MBko littermates at 20 weeks of age. Our data shed novel insights into MB's role in linking oxygen and lipid-based thermogenic metabolism. The findings suggest potential new strategies of targeting the MB pathway to treat metabolic disorders related to diminishing energy expenditure.

Metabolic responses to hypoxia play important roles in cell survival strategies and disease pathogenesis in humans. However, the homeostatic adjustments that balance changes in energy supply and demand to maintain organismal function under chronic low oxygen conditions remain incompletely understood, making it difficult to distinguish adaptive from maladaptive responses in hypoxia-related pathologies. We integrated metabolomic and proteomic profiling with mitochondrial respirometry and blood gas analyses to comprehensively define the physiological responses of skeletal muscle energy metabolism to 16 days of high-altitude hypoxia (5260 m) in healthy volunteers from the AltitudeOmics project. In contrast to the view that hypoxia down-regulates aerobic metabolism, results show that mitochondria play a central role in muscle hypoxia adaptation by supporting higher resting phosphorylation potential and enhancing the efficiency of long-chain acylcarnitine oxidation. This directs increases in muscle glucose toward pentose phosphate and one-carbon metabolism pathways that support cytosolic redox balance and help mitigate the effects of increased protein and purine nucleotide catabolism in hypoxia. Muscle accumulation of free amino acids favor these adjustments by coordinating cytosolic and mitochondrial pathways to rid the cell of excess nitrogen, but might ultimately limit muscle oxidative capacity in vivo. Collectively, these studies illustrate how an integration of aerobic and anaerobic metabolism is required for physiological hypoxia adaptation in skeletal muscle, and highlight protein catabolism and allosteric regulation as unexpected orchestrators of metabolic remodeling in this context. These findings have important implications for the management of hypoxia-related diseases and other conditions associated with chronic catabolic stress.

Intracellular signaling pathways not only control cell proliferation and survival, but also regulate the provision of cellular energy and building blocks through mitochondrial and non-mitochondrial metabolism. Wild-type and oncogenic RAF kinases have been shown to prevent apoptosis following the removal of interleukin 3 (IL-3) from mouse pro-myeloid 32D cells by reducing mitochondrial reactive oxygen species production. To study primary effects of RAF on mitochondrial energy metabolism, we applied high-resolution respirometry after short-term IL-3 deprivation (8 h), before 32D cells show detectable signs of cell death. Respiration in intact 32D cells was suppressed as an early event following removal of IL-3, but remained more stable in 32D cells expressing the v-RAF oncogene. In permeabilized 32D cells deprived of IL-3, respiratory capacities of the NADH-pathway, the convergent NADH&succinate-pathway, and Complex IV activity were decreased compared to cells grown in the presence of IL-3, whereas succinate-supported respiration remained unchanged, consistent with control by Complex IV. The apparent Complex IV excess capacity was zero above NADH&succinate-pathway capacity reconstituting tricarboxylic acid cycle function. In comparison, electron flow reached only 60% when supported by succinate alone through Complexes II, III and IV, and was therefore relatively insensitive to Complex IV injuries up to a threshold of 40% inhibition. A slight increase in respiration following addition of cytochromec, a marker of mitochondrial outer membrane leakage, was present after IL-3 depletion, indicating mitochondrial fragility. Our results highlight a novel link between the key mitogenic and survival kinase CRAF and mitochondrial energy homeostasis.

The number of studies on mitochondrial function is growing as a result of the recognition of the pivotal role of an intact mitochondrial function in numerous diseases. Measurements of oxygen consumption by the mitochondria in human skeletal muscle are used in many studies. There are several advantages of studying mitochondrial respiration in permeabilized fibers (Pfi), but the method requires a manual procedure of mechanical separation of the fiber bundles in the biopsy and chemical permeabilization of the cell membrane. This is time-consuming and subject to interpersonal variability. An alternative is to use a semiautomatic tool for preparation of a homogenate of the muscle biopsy. We investigated whether the PBI shredder is useful in preparing a muscle homogenate for measurements of mitochondrial respiratory capacity. The homogenate is compared with the Pfi preparation. Maximal respiratory capacity was significantly reduced in the homogenate compared with the Pfi from human skeletal muscle. A marked cytochrome c response was observed in the homogenate, which was not the case with the Pfi, indicating that the outer mitochondrial membrane was not intact. The mitochondria in the homogenate were more uncoupled compared with the Pfi. Manual permeabilization is an advantageous technique for preparing human skeletal muscle biopsies for respirometry.

At the annual Iditarod Race, Alaskan Huskies repeatedly run for up to 8 hours at 16 km/h to complete 1600 km. We previously demonstrated high rates of mitochondrial protein synthesis in Alaskan Huskies, which we suspected allowed rapid remodeling of mitochondrial proteins in response to energetic stress. The purpose of this study was to examine mitochondrial respiration in permeabilized skeletal muscle fibers of Alaskan Huskies in the offseason (Non-raced) and following the 1600 km Iditarod Sled Dog Race (Raced). We hypothesized that compared to Non-raced Huskies, raced Huskies that completed a 1600 km race would have greater mitochondrial respiratory capacities, and improvements in capacities of oxidative phosphorylation (OXPHOS) based on NADH-generating substrates as compared to fatty acids. Using high-resolution respirometry (HRR) we investigated the respiration of permeabilized muscle fibers from Alaskan Huskies. Maximum capacities were 254±26 pmol.s-1.mg-1 for OXPHOS (coupled, P) and 254±37 pmol.s-1.mg-1 for the electron transfer system (ETS; non-coupled, E). After racing respiratory capacities from NADH-linked substrates, but not fat-derived substrates increased. Finally, the OXPHOS to ETS capacity ratio (P/E) increased after racing from 0.90±0.03 to 0.97±0.02. From our previous studies and the current study, we conclude that Alaskan Huskies maintain high mitochondrial protein turnover to facilitate rapid adaptation to environmental extremes and energetic challenges.

Heart failure is a consequence of progressive deterioration of cardiac performance. Little is known about the role of impaired oxidative phosphorylation in the progression of the disease, since previous studies of mitochondrial injuries are restricted to end-stage chronic heart failure. The present study aimed at evaluating the involvement of mitochondrial dysfunction in the development of human heart failure. We measured the control of oxidative phosphorylation with high-resolution respirometry in permeabilized myocardial fibres from donor hearts (controls), and patients with no or mild heart failure but presenting with heart disease, or chronic heart failure due to dilated or ischemic cardiomyopathy. The capacity of the phosphorylation system exerted a strong limitation on oxidative phosphorylation in the human heart, estimated at 121 pmol O(2)s(-1)mg(-1) in the healthy left ventricle. In heart disease, a specific defect of the phosphorylation system, Complex I-linked respiration, and mass-specific fatty acid oxidation were identified. These early defects were also significant in chronic heart failure, where the capacities of the oxidative phosphorylation and electron transfer systems per cardiac tissue mass were decreased with all tested substrate combinations, suggesting a decline of mitochondrial density. Oxidative phosphorylation and electron transfer system capacities were higher in ventricles compared to atria, but the impaired mitochondrial quality was identical in the four cardiac chambers of chronic heart failure patients. Coupling was preserved in heart disease and chronic heart failure, in contrast to the mitochondrial dysfunction observed after prolonged cold storage of cardiac tissue. Mitochondrial defects in the phosphorylation system, Complex I respiration and mass-specific fatty acid oxidation occurred early in the development of heart failure. Targeting these mitochondrial injuries with metabolic therapy may offer a promising approach to delay the progression of heart disease.

Recently, immunohistochemical analysis of myoglobin (MB) in human breast cancer specimens has revealed a surprisingly widespread expression of MB in this nonmuscle context. The positive correlation with hypoxia-inducible factor 2α (HIF-2α) and carbonic anhydrase IX suggested that oxygen regulates myoglobin expression in breast carcinomas. Here, we report that MB mRNA and protein levels are robustly induced by prolonged hypoxia in breast cancer cell lines, in part via HIF-1/2-dependent transactivation. The hypoxia-induced MB mRNA originated from a novel alternative transcription start site 6 kb upstream of the ATG codon. MB regulation in normal and tumor tissue may thus be fundamentally different. Functionally, the knockdown of MB in MDA-MB468 breast cancer cells resulted in an unexpected increase of O2 uptake and elevated activities of mitochondrial enzymes during hypoxia. Silencing of MB transcription attenuated proliferation rates and motility capacities of hypoxic cancer cells and, surprisingly, also fully oxygenated breast cancer cells. Endogenous MB in cancer cells is apparently involved in controlling oxidative cell energy metabolism, contrary to earlier findings on mouse heart, where the targeted disruption of the Mb gene did not effect myocardial energetics and O2 consumption. This control function of MB seemingly impacts mitochondria and influences cell proliferation and motility, but it does so in ways not directly related to the facilitated diffusion or storage of O2. Hypothetically, the mitochondrion-impairing role of MB in hypoxic cancer cells is part of a novel tumor-suppressive function.

Breast cancer cells can survive and proliferate under harsh conditions of nutrient deprivation, including limited oxygen and glucose availability. We hypothesized that such environments trigger metabolic adaptations of mitochondria, which promote tumor progression. Here, we mimicked aglycemia and hypoxia in vitro and compared the mitochondrial and cellular bioenergetic adaptations of human breast cancer (HTB-126) and non-cancer (HTB-125) cells that originate from breast tissue. Using high-resolution respirometry and western blot analyses, we demonstrated that 4 days of glucose deprivation elevated oxidative phosphorylation five-fold, increased the spread of the mitochondrial network without changing its shape, and decreased the apparent affinity of oxygen in cancer cells (increase in C 50 ), whereas it remained unchanged in control cells. The substrate control ratios also remained constant following adaptation. We also observed the Crabtree effect, specifically in HTB-126 cells. Likewise, sustained hypoxia (1% oxygen during 6 days) improved cell respiration in non-cancer cells grown in glucose or glucose-deprived medium (+ 32% and +38%, respectively). Conversely, under these conditions of limited oxygen or a combination of oxygen and glucose deprivation for 6 days, routine respiration was strongly reduced in cancer cells (−36% in glucose medium, −24% in glucose-deprived medium). The data demonstrate that cancer cells behave differently than normal cells when adapting their bioenergetics to microenvironmental conditions. The differences in hypoxia and aglycemia tolerance between breast cancer cells and non-cancer cells may be important when optimizing strategies for the treatment of breast cancer.

Maximal ADP-stimulated mitochondrial respiration depends on convergent electron flow through Complexes I+II to the Q-junction of the electron transport system (ETS). In most studies of respiratory control in mitochondrial preparations, however, respiration is limited artificially by supplying substrates for electron input through either Complex I or II. High-resolution respirometry with minimal amounts of tissue biopsy (1-3mg wet weight of permeabilized muscle fibres per assay) provides a routine approach for multiple substrate-uncoupler-inhibitor titrations. Under physiological conditions, maximal respiratory capacity is obtained with glutamate+malate+succinate, reconstituting the operation of the tricarboxylic acid cycle and preventing depletion of key metabolites from the mitochondrial matrix. In human skeletal muscle, conventional assays with pyruvate+malate or glutamate+malate yield submaximal oxygen fluxes at 0.50-0.75 of capacity of oxidative phosphorylation (OXPHOS). Best estimates of muscular OXPHOS capacity at 37 degrees C (pmol O(2)s(-1)mg(-1) wet weight) with isolated mitochondria or permeabilized fibres, suggest a range of 100-150 and up to 180 in healthy humans with normal body mass index and top endurance athletes, but reduction to 60-120 in overweight healthy adults with predominantly sedentary life style. The apparent ETS excess capacity (uncoupled respiration) over ADP-stimulated OXPHOS capacity is high in skeletal muscle of active and sedentary humans, but absent in mouse skeletal muscle. Such differences of mitochondrial quality in skeletal muscle are unexpected and cannot be explained at present. A comparative database of mitochondrial physiology may provide the key for understanding the functional implications of mitochondrial diversity from mouse to man, and evaluation of altered mitochondrial respiratory control patterns in health and disease.

Altered mitochondrial metabolism plays a pivotal role in the development and progression of various diseases, including cancer. Cell lines are frequently used as models to study mitochondrial (dys)function, but little is known about their mitochondrial respiration and metabolic properties in comparison to the primary tissue of origin. We have developed a method for assessment of oxidative phosphorylation in prostate tissue samples of only 2 mg wet weight using high-resolution respirometry. Reliable protocols were established to investigate the respiratory activity of different segments of the mitochondrial electron transfer system (ETS) in mechanically permeabilized tissue biopsies. Additionally, the widely used immortalized prostate epithelial and fibroblast cell lines, RWPE1 and NAF, representing the major cell types in prostate tissue, were analyzed and compared to the tissue of origin. Our results show that mechanical treatment without chemical permeabilization agents or sample processing constitutes a reliable preparation method for OXPHOS analysis in small amounts of prostatic tissue typically obtained by prostate biopsy. The cell lines represented the bioenergetic properties of fresh tissue to a limited extent only. Particularly, tissue showed a higher oxidative capacity with succinate and glutamate, whereas pyruvate was a substrate supporting significantly higher respiratory activities in cell lines. Several fold higher zinc levels measured in tissue compared to cells confirmed the role of aconitase for prostate-specific metabolism in agreement with observed respiratory properties. In conclusion, combining the flexibility of cell culture models and tissue samples for respirometric analysis are powerful tools for investigation of mitochondrial function and tissue-specific metabolism.

Alterations in mitochondrial function are believed to play a major role in aging processes in many species, including fungi and animals, and increased oxidative stress is considered a major consequence of altered mitochondrial function. In support of this theory, a lot of correlative evidence has been collected, suggesting that changes in mitochondrial DNA accumulate with age in certain tissues. Furthermore, genetic experiments from lower eukaryotic model organisms, indicate a strong correlative link between increased resistance to oxidative stress and an extended lifespan; in addition, limited experimental evidence suggests that the inhibition of mitochondrial function by selected pharmacologically active compounds can extend lifespan in certain species. However, changes in mitochondrial function may affect aging in a different way in various tissues, and a clear statement about the role of mitochondrial deterioration during physiological aging is missing for most if not all species. At this point, respirometric analyses of mitochondrial function provide a tool to study age-associated changes in mitochondrial respiratory chain function and mitochondrial ATP production within living cells and isolated mitochondria. In the recent years, new instruments have been developed, which allow for an unprecedented high-resolution respirometry, which enables us to determine many parameters of mitochondrial function in routine assays using small samples of biological material. It is conceivable that this technology will become an important tool for all those, who are interested in experimentally addressing the mitochondrial theory of aging. In this article, we provide a synopsis of traditional respirometry and the advances of modern high-resolution respirometry, and discuss how future applications of this technology to recently established experimental models in aging research may provide exciting new insights into the role of mitochondria in the aging process.

Background. Protection of vascular endothelium is a critical factor in organ preservation for transplantation. This study aims at a morphological assessment of endothelial cell injury in a comparison of storage solutions, using a cell culture model of cold preservation and rewarming. Materials and methods. Human umbilical vein endothelial cells (HUVEC) were cultured in monolayer and exposed to hypothermic storage in University of Wisconsin (UW), histidine‐tryptophane‐ ketoglutarate (HTK), and EuroCollins solutions for 6 h and subsequent rewarming for 30 min or 6 h. Alterations of subcellular structures and cell‐ cell contacts were analyzed by transmission electron microscopy (TEM) and light microscopic assessment after actin and nuclear staining. Results. Structural alterations of mitochondria, endoplasmic reticulum, nuclei, and cytoskeletal fibers as well as disruption of intercellular contacts were found after cold storage in HTK and EuroCollins solutions. In contrast, storage in UW solution resulted in minimum changes of stress fibers only. A rapid rearrangement of structural alterations was achieved during rewarming in cell culture medium in all experimental groups. Conclusions. Preservation of endothelial cell structure is best achieved by UW solution. Ultrastructural cell damage is a direct consequence of hypothermic storage and is fully reversible during rewarming after short storage times. © 1999 Academic Press

Oxygen levels in the intracellular microenvironment of tissues such as heart are extremely low, at 1–2% of standard atmospheric oxygen pressure. Kinetic studies with isolated mitochondria suggest a regulatory role of oxygen under these conditions, particularly in active states at high ADP concentration, when oxygen affinity was lower than in the resting state at ADP limitation. The oxygen pressure at 50% of maximum flux, p50, was 0.035 and 0.057 kPa in heart and liver mitochondria, respiring in state 3 on substrates for complex I or II and II, respectively. p50 in the resting state 4 was 0.02 kPa. The apparent kinetic efficiency, Jmax/p50, increased from the resting to the active state, despite the decrease of oxygen affinity, 1/p50. Consequently, the relative increase of respiratory flux by ADP activation, expressed as the adenylate control ratio, declined under hypoxia, but not to the extreme of a complete loss of the scope for activation, which would occur at constant Jmax/p50. High oxygen affinity is achieved by an excess capacity of cytochrome c oxidase relative to the respiratory chain and a correspondingly low turnover rate of this enzyme, consistent with the concept of kinetic trapping of oxygen [1].

We studied the oxygen dependence of respiration in cultured human umbilical vein endothelial cells by use of high-resolution respirometry. The rate of oxygen consumption varied from 30 to 50 pmol O2.s-1.(10(6) cells)-1 over a sixfold range of cell densities. Respiration was stimulated up to 3.5-fold by uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone or 2,4-dinitrophenol, and the PO2 at half-maximal respiration (P50) increased from 0.05 to 0.12 kPa (0.3 to 0.9 Torr) with respiratory rate. P50 decreased to a minimum of 0.02 kPa when uncoupled cells were inhibited to control levels. Differences in cell size explained a variation of approximately 0.015 kPa in P50 at similar respiratory rates per cell. Oxygen diffusion to mitochondria contributed maximally 30% to the regulation of P50 in coupled cells, as deduced from the shallow slope of the flux dependence of P50 in uncoupled-inhibited cells compared with the slope in coupled cells. Therefore 70% of the flux dependence of P50 in coupled cells was caused by changes in metabolic state, which correlated with respiratory rate.

A review with 48 refs. The topics discussed include mass and energy balances for open and non-steady state systems, calcn. of reaction enthalpies, and enthalpy balance in aerobic and anaerobic growth. [on SciFinder (R)]

Across a wide range of species and body mass a close matching exists between maximal conductive oxygen delivery and mitochondrial respiratory rate. In this study we investigated in humans how closely in-vivo maximal oxygen consumption (VO(2) max) is matched to state 3 muscle mitochondrial respiration. High resolution respirometry was used to quantify mitochondrial respiration from the biopsies of arm and leg muscles while in-vivo arm and leg VO(2) were determined by the Fick method during leg cycling and arm cranking. We hypothesized that muscle mitochondrial respiratory rate exceeds that of systemic oxygen delivery. The state 3 mitochondrial respiration of the deltoid muscle (4.3±0.4 mmol o(2)kg(-1) min(-1)) was similar to the in-vivo VO(2) during maximal arm cranking (4.7±0.5 mmol O(2) kg(-1) min(-1)) with 6 kg muscle. In contrast, the mitochondrial state 3 of the quadriceps was 6.9±0.5 mmol O(2) kg(-1) min(-1), exceeding the in-vivo leg VO(2) max (5.0±0.2 mmol O(2) kg(-1) min(-1)) during leg cycling with 20 kg muscle (P

Nitric oxide (NO) inhibits mitochondrial respiration by decreasing the apparent affinity of cytochrome c oxidase (CcO) for oxygen. Using iNOS-transfected HEK 293 cells to achieve regulated intracellular NO production, we determined NO and O(2) concentrations and mitochondrial O(2) consumption by high-resolution respirometry over a range of O(2) concentrations down to nanomolar. Inhibition of respiration by NO was reversible, and complete NO removal recovered cell respiration above its routine reference values. Respiration was observed even at high NO concentrations, and the dependence of IC(50) on [O(2)] exhibits a characteristic but puzzling parabolic shape; both these features imply that CcO is protected from complete inactivation by NO and are likely to be physiologically relevant. We present a kinetic model of CcO inhibition by NO that efficiently predicts experimentally determined respiration at physiological O(2) and NO concentrations and under hypoxia, and accurately predicts the respiratory responses under hyperoxia. The model invokes competitive and uncompetitive inhibition by binding of NO to the reduced and oxidized forms of CcO, respectively, and suggests that dissociation of NO from reduced CcO may involve its O(2)-dependent oxidation. It also explains the non-linear dependence of IC(50) on O(2) concentration, and the hyperbolic increase of c(50) as a function of NO concentration.

The peritoneal mesothelium acts as a regulator of serosal responses to injury, infection, and neoplastic diseases. After inflammation of the serosal surfaces, proinflammatory cytokines induce an "activated" mesothelial cell phenotype, the mitochondrial aspect of which has not previously been studied. After incubation of cultured human peritoneal mesothelial cells with interleukin (IL)-1beta for 48 h, respiratory activity of suspended cells was analyzed by high-resolution respirometry. Citrate synthase (CS) and lactate dehydrogenase (LDH) activities were determined by spectrophotometry. Treatment with IL-1beta resulted in a significant decline of respiratory capacity (p < 0.05). Respiratory control ratios (i.e., uncoupled respiration at optimum carbonyl cyanide p-trifluoromethoxyphenylhydrazone concentration divided by oligomycin inhibited respiration measured in unpermeabilized cells) remained as high as 11, indicating well-coupled mitochondria and functional integrity of the inner mitochondrial membrane. Whereas respiratory capacities of the cells declined in proportion with decreased CS activity (p < 0.05), LDH activity increased (p < 0.05). Taken together, these results indicate that IL-1beta exposure of peritoneal mesothelial cells does not lead to irreversible defects or inhibition of specific components of the respiratory chain, but is associated with a decrease of mitochondrial content of the cells that is correlated with an increase in LDH (and thus glycolytic) capacity.

Mutations in the gene SURF1 prevent synthesis of cytochrome- c oxidase (COX)-specific assembly protein and result in a fatal neurological disorder, Leigh syndrome. Because this severe COX deficiency presents with barely detectable changes of cellular respiratory rates under normoxic conditions, we analyzed the respiratory response to low oxygen in cultured fibroblasts harboring SURF1 mutations with high-resolution respirometry. The oxygen kinetics was quantified by the partial pressure of oxygen (Po2) at half-maximal respiration rate (P50) in intact coupled cells and in digitonin-permeabilized uncoupled cells. In both cases, the P50in patients was elevated 2.1- and 3.3-fold, respectively, indicating decreased affinity of COX for oxygen. These results suggest that at physiologically low intracellular Po2, the depressed oxygen affinity may lead in vivo to limitations of respiration, resulting in impaired energy provision in Leigh syndrome patients.

Mitochondrial respiratory function was studied in permeabilized pig liver biopsies. The cell membrane was permeabilized mechanically in tissue samples of 2–7 mg, for application of a standardized substrate/inhibitor titration protocol in high-resolution respirometry. Specific respirometric tests demonstrated complete plasma membrane permeabilization and accessibility of substrates to intact mitochondria. High respiratory adenylate control ratios and cytochrome c conservation in the tissue preparation were comparable or even better than in isolated mitochondria. Citrate synthase and cytochrome c oxidase activities remained at 85% of controls after up to 98 h storage of liver tissue at 0°C in histidine–tryptophan–ketoglutarate solution. Multiple mitochondrial defects, however, were indicated after 48 h cold storage by the decline in respiratory capacity, which was lowered to a larger extent with complex I substrates compared to respiration with substrates for complex II or IV, measured in the absence of cytochrome c. After prolonged ischemia, the adenylate control ratio was significantly reduced, and cytochrome c depletion was detected by the stimulatory effect of cytochrome c. High-resolution respirometry allows the assessment of mitochondrial function in a few milligrams of permeabilized liver tissue, without isolation of mitochondria. This provides a basis for the analysis of mitochondrial function in human liver biopsies.

Questions as to the critical stress factor and primary targets of cold ischemia/reperfusion (CIR) injury were addressed by comparing mitochondrial defects caused by (1) CIR injury and (2) intracellular Ca2+ overload. CIR was simulated in transformed human umbilical vein endothelial cell cultures (tEC) by 8 h cold anoxia in University of Wisconsin solution and reoxygenation at 37 degrees C. Intracellular Ca2+ concentrations were changed by permeabilization of suspended cells with digitonin in culture medium (RPMI, 0.4 mM Ca2+). Binding of free Ca2+ by ethylene glycol-bis(beta-aminoethylether)-N,N,N',N'-tetraacetic acid in RPMI or mitochondrial incubation medium served as controls. Extracellular Ca2+ protected the cell membrane against permeabilization. Mitochondrial functions were determined before and after permeabilization of the cell membrane. After CIR, mitochondrial respiratory capacity declined, but oxygen consumption remained coupled to adenosine triphosphate (ATP) production. In contrast, Ca2+ overload caused uncoupling of mitochondrial respiration. High intracellular Ca2+ overload, therefore, does not reproduce cold ischemia/reperfusion injury in endothelial cells.

Development of hibernation strategies for cold preservation of human organs represents a far-reaching goal in transplantation surgery. Short cold storage times of

Iron modulates the expression of the critical citric acid cycle enzyme aconitase via a translational mechanism involving iron regulatory proteins. Thus, the present study was undertaken to investigate the consequences of iron perturbation on citric acid cycle activity, oxidative phosphorylation and mitochondrial respiration in the human cell line K-562. In agreement with previous data iron increases the activity of mitochondrial aconitase while it is reduced upon addition of the iron chelator desferrioxamine (DFO). Interestingly, iron also positively affects three other citric acid cycle enzymes, namely citrate synthase, isocitric dehydrogenase, and succinate dehydrogenase, while DFO decreases the activity of these enzymes. Consequently, iron supplementation results in increased formation of reducing equivalents (NADH) by the citric acid cycle, and thus in increased mitochondrial oxygen consumption and ATP formation via oxidative phosphorylation as shown herein. This in turn leads to downregulation of glucose utilization. In contrast, all these metabolic pathways are reduced upon iron depletion, and thus glycolysis and lactate formation are significantly increased in order to compensate for the decrease in ATP production via oxidative phosphorylation in the presence of DFO. Our results point to a complex interaction between iron homeostasis, oxygen supply and cellular energy metabolism in human cells.

Endothelial activation is a central feature of preservation-induced allograft injury. The present study aims at a quantitative assessment of stress proteins, adhesion molecules, and interleukin-8 in a cell culture-based model of organ preservation. Human umbilical vein endothelial cells were exposed to cold, hypoxic storage in University of Wisconsin (UW), histidine-tryptophane-ketoglutarate (HTK), and EuroCollins solutions for 8 h with subsequent rewarming/reoxygenation (rew/reox) for 1 and 4 h. A cell-based ELISA was designed for detection of heat shock proteins (HSP) 60 and 70, intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelial leukocyte adhesion molecule-1 (ELAM-1). Immunohistochemical staining was performed for comparison. Interleukin-8 was quantified by ELISA. HSP 70 was expressed after cold storage in HTK and EuroCollins solution and after rew/reox in all groups. A constitutive expression of HSP 60 was observed with further upregulation after rew/reox following cold storage in all experimental groups. ICAM-1 was clearly upregulated, but VCAM-1 showed only weak expression after cold storage and rew/reox. ELAM-1 was detectable in minimal amounts after cold storage but was considerably upregulated after 4 h of rew/reox. A significant increase of interleukin-8 release could be found after 4 h of rew/reox following storage in EuroCollins solution. Expression of stress proteins can be considered as a new parameter of preservation-associated endothelial activation. Apart from possible protective effects, allograft vasculopathy could be in part a consequence of the antigeneic potential of heat shock proteins connected with effects caused by adhesion molecules and inflammatory cytokines.

0 RGAN PRESERVATION under hypothermic ischemia is enhanced by storage solutions that protect the vascular endothelium from ischemia-reperfusion injury. Ischemia-reperfusion injury leads to primary graft failure and chronic rejection, and is commonly assessed by measuring endothelial activation and damage of the endothelial plasma membrane. However, corresponding primary intracellular events are little understood compared with the secondary cytokine/adhesion molecule cascade and inflammatory responses.‘-* Because protection of intracellular and cell membrane function is fundamental for further improvement of organ preservation, we developed highresolution respirometry as a sensitive diagnostic test for mitochondrial and plasma membrane competence.3 Whereas the plasma membrane remained impermeable after clinically relevant cold storage times of 8 hours and 20