Measure Of Mitochondrial Function Research Articles

Measurement of Mitochondrial Function in Human Platelets

Measurement of Mitochondrial Function in Human Platelets

Assessing bioenergetic function in response to oxidative stress by metabolic profiling

It is now clear that mitochondria are an important target for oxidative stress in a broad range of pathologies, including cardiovascular disease, diabetes, neurodegeneration, and cancer. Methods for assessing the impact of reactive species on isolated mitochondria are well established but constrained by the need for large amounts of material to prepare intact mitochondria for polarographic measurements. With the availability of high-resolution polarography and fluorescence techniques for the measurement of oxygen concentration in solution, measurements of mitochondrial function in intact cells can be made. Recently, the development of extracellular flux methods to monitor changes in oxygen concentration and pH in cultures of adherent cells in multiple-sample wells simultaneously has greatly enhanced the ability to measure bioenergetic function in response to oxidative stress. Here we describe these methods in detail using representative cell types from renal, cardiovascular, nervous, and tumorigenic model systems while illustrating the application of three protocols to analyze the bioenergetic response of cells to oxidative stress.

Retinal flavoprotein fluorescence correlates with mitochondrial stress, apoptosis, and chemokine expression

Oxidative stress and mitochondrial dysfunction occur before apoptosis in many retinal diseases. Under these conditions, a larger fraction of flavoproteins become oxidized and, when excited by blue-light, emit green flavoprotein fluorescence (FPF). In this study, we evaluated the utility of FPF as an early indicator of mitochondrial stress, pre-apoptotic cellular instability, and apoptosis of human retinal pigment epithelial (HRPE) cells subjected to hydrogen peroxide (H 2O 2) or monocytes (unstimulated or interferon-γ-stimulated) in vitro and of freshly-isolated pieces of human and rat neural retina subjected to H 2O 2 ex vivo. Increased FPF of HRPE cells exposed to H 2O 2 correlated with reduced mitochondrial membrane potential (ΔΨm) and increased apoptosis in a time- and dose-dependent manner. HRPE cells co-cultured with monocytes had increased FPF that correlated in a time-dependent manner with reduced ΔΨm, increased apoptosis, and early expression of pro-inflammatory chemokines, interleukin-8 (IL8) and monocyte chemotactic factor-1 (MCP1), which are known to be induced by oxidative stress. Increased FPF, reduced ΔΨm, and upregulation of IL8 and MCP1 occurred as early as 1–2 h after exposure to stressors, while apoptosis did not occur in HRPE cells until later time points. The antioxidant, N-acetyl-cysteine (NAC), inhibited increased FPF and apoptosis of HRPE cells subjected to H 2O 2. Increased FPF of human and rat neural retina also correlated with increased apoptosis. This study suggests that FPF is a useful measure of mitochondrial function in retinal cells and tissues and can detect early mitochondrial dysfunction that may precede apoptosis.

Measurement of mitochondrial function in equine blood lymphocytes

The morphopathological features of “equine atypical myopathy” are consistent with a primary mitochondrial disease but this hypothesis hasn’t been confirmed yet. The cationic dye JC-1 exhibits potential-dependent accumulation in mitochondria that is detectable by a fluorescence shift from green to orange. So, mitochondrial membrane potential can be measured by the orange/green fluorescence intensity ratio. This ratio has been already used as an indicator of mitochondrial potential in isolated mitochondria but also in intact cells (spermatozoa, myocytes, neurons). The purpose of this study was to obtain reference values of mitochondrial function in lymphocytes of healthy horses by a flow cytometric standardized analytic procedure. This assay will prove to be useful if and only if the mitochondrial muscular dysfunction is the consequence of a polysystemic mitochondrial disease that affects circulating leucocytes too. A group of 31 horses clinically normal were enrolled into the study. Results showed a great stability of the JC-1 fluorescence lymphocytes values over time, their independence of age and sex and their hypersensitivity to intoxication with a mitochondrial potential dissipator. So, whenever a quantitative measurement of mitochondrial function in a horse is desired, blood samples should be taken in sodium citrate tubes and kept at room temperature for a maximum of 5 hours before the beginning of laboratory procedure.

Reply to Padilla, Hamilton, Lundgren, Mckenzie, and Mickleborough

LETTERS TO THE EDITORReply to Padilla, Hamilton, Lundgren, Mckenzie, and MickleboroughJoffrey Zoll, Elodie Ponsot, Stephane Dufour, and Martin FlückJoffrey Zoll, Elodie Ponsot, Stephane Dufour, and Martin FlückPublished Online:01 Aug 2007https://doi.org/10.1152/japplphysiol.00535.2007MoreSectionsPDF (112 KB)Download PDF ToolsExport citationAdd to favoritesGet permissionsTrack citations ShareShare onFacebookTwitterLinkedInEmailWeChat To the Editor: We appreciate the interest of Padilla et al. (3) in our results showing the effect of a modified intermittent hypoxic training (IHT) in endurance athletes. Although the authors raise several comments, the main scientific question seems to concern the rationale to investigate the transcript level for carbonic anhydrase 3 (CA3) in the skeletal muscles of our athletes.To clarify this issue, it is worth bearing in mind that the study was not specifically performed to identify muscular markers of hypoxia exposure. Rather, the objective was to highlight adaptations of athletes' skeletal muscle after performing specific hypoxia training sessions. We did not, therefore, primarily intend to describe the effects of chronic or acute hypoxia alone, as in the two studies cited by Padilla et al. Consequently, we explored the respective muscular transcript levels of genes involved in mitochondrial biogenesis, redox regulation, and glucose uptake to ascertain whether they could participate in the improvement of endurance performance following 6-wk IHT.We chose to trace mRNA of CA3 because this enzyme is considered as a potential factor of mitochondrial flux improvement and because high cytosolic concentrations of CA3 have been reported in mammalian skeletal muscle type I (slow-oxidative fibers; Ref. 1). Its RNA level also showed significant plasticity with muscle atrophy and reloading (2). Although our data point out an involvement of CA3 to the hypoxia adaptation of muscle, there is certainly a need to study other carbonic anhydrase isoforms, which could play a role in the muscular adaptations following IHT.Nevertheless, we thank Padilla et al. for their thorough review of our papers, which gives us the opportunity to explain some of their concerns and to correct one mistake that unfortunately appeared on Fig. 2 in the third part of the trilogy.1) A total of 18 volunteered subjects completed the research protocol (6-wk training program, pre- and post-training muscle biopsies of the vastus lateralis muscle and treadmill performance evaluations). As regularly reported in invasive human studies, biopsies did not always give us enough biological material to carry out the measurements of mitochondrial function and mRNAs expression, explaining why the population decline to 15 subjects in the second and third part of our work (4, 5).2) By not specifying the interaction effect, our aim was to simplify the result sections of the papers, which already report an important amount of data.3) The range of values for Tlim in the printed Fig. 2 does not make sense as presented. We apologize for this mistake we did not notice and provide the exact, double-checked, correlations with the actual range of values.In conclusion, CA3 is possibly not the best marker of hypoxia exposure, but we demonstrate that this isoenzyme of CA is specifically upregulated in hypoxia-trained subjects and presumably plays a role, together with other factors, in the ultimate improvement of endurance performance capacity. Download figureDownload PowerPointREFERENCES1 Geers C, Gros G. Carbon dioxide transport and carbonic anhydrase in blood and muscle. Physiol Rev 80: 681–715, 2000.Link | ISI | Google Scholar2 Fluck M, Schmutz S, Wittwer M, Hoppeler H, Desplanches D. Transcriptional reprogramming during reloading of atrophied rat soleus muscle. Am J Physiol Regul Integr Comp Physiol 289: R4–R14, 2005.Link | ISI | Google Scholar3 Padilla J, Hamilton S, Lundgren E, Mckenzie J, Mickleborough T. Exercise training in normobaric hypoxia: is carbonic anyhdrase III the best marker of hypoxia? J Appl Physiol. doi:10.1152/japplphysiol.00408.2007.Google Scholar4 Ponsot E, Dufour SP, Zoll J, Doutrelau S, N'Guessan B, Geny B, Hoppeler H, Lampert E, Mettauer B, Ventura-Clapier R, Richard R. Exercise training in normobaric hypoxia in endurance runners. II. Improvement of mitochondrial properties in skeletal muscle. J Appl Physiol 100: 1249–1257, 2006.Link | ISI | Google Scholar5 Zoll J, Ponsot E, Dufour S, Doutreleau S, Ventura-Clapier R, Vogt M, Hoppeler H, Richard R, Fluck M. Exercise training in normobaric hypoxia in endurance runners. III. Muscular adjustments of selected gene transcripts. J Appl Physiol 100: 1258–1266, 2006.Link | ISI | Google ScholarAUTHOR NOTESAddress for reprint requests and other correspondence: J. Zoll, Institut de Physiologie, Faculté de Médecine, 4, rue Kirschleger, F-67085 Strasbourg Cedex, France (e-mail: [email protected]) Download PDF Previous Back to Top FiguresReferencesRelatedInformation More from this issue > Volume 103Issue 2August 2007Pages 731-732 Copyright & PermissionsCopyright © 2007 the American Physiological Societyhttps://doi.org/10.1152/japplphysiol.00535.2007History Published online 1 August 2007 Published in print 1 August 2007 Metrics

Intersubject differences in the effect of acidosis on phosphocreatine recovery kinetics in muscle after exercise are due to differences in proton efflux rates

(31)P magnetic resonance spectroscopy provides the possibility of obtaining bioenergetic data during skeletal muscle exercise and recovery. The time constant of phosphocreatine (PCr) recovery (tau(PCr)) has been used as a measure of mitochondrial function. However, cytosolic pH has a strong influence on the kinetics of PCr recovery, and it has been suggested that tau(PCr) should be normalized for end-exercise pH. A general correction can only be applied if there are no intersubject differences in the pH dependence of tau(PCr). We investigated the pH dependence of tau(PCr) on a subject-by-subject basis. Furthermore, we determined the kinetics of proton efflux at the start of recovery. Intracellular acidosis slowed PCr recovery, and the pH dependence of tau(PCr) differed among subjects, ranging from -33.0 to -75.3 s/pH unit. The slope of the relation between tau(PCr) and end-exercise pH was positively correlated with both the proton efflux rate and the apparent proton efflux rate constant, indicating that subjects with a smaller pH dependence of tau(PCr) have a higher proton efflux rate. Our study implies that simply correcting tau(PCr) for end-exercise pH is not adequate, in particular when comparing patients and control subjects, because certain disorders are characterized by altered proton efflux from muscle fibers.

Cerebral energetic effects of creatine supplementation in humans

There has been considerable interest in the use of creatine (Cr) supplementation to treat neurological disorders. However, in contrast to muscle physiology, there are relatively few studies of creatine supplementation in the brain. In this report, we use high-field MR (31)P and (1)H spectroscopic imaging of human brain with a 7-day protocol of oral Cr supplementation to examine its effects on cerebral energetics (phosphocreatine, PCr; ATP) and mitochondrial metabolism (N-acetyl aspartate, NAA; and Cr). We find an increased ratio of PCr/ATP (day 0, 0.80 +/- 0.10; day 7, 0.85 +/- 09), with this change largely due to decreased ATP, from 2.7 +/- 0.3 mM to 2.5 +/- 0.3 mM. The ratio of NAA/Cr also decreased (day 0, 1.32 +/- 0.17; day 7 1.18 +/- 0.13), primarily from increased Cr (9.6 +/- 1.9 to 10.1 +/- 2.0 mM). The Cr-induced changes significantly correlated with the basal state, with the fractional increase in PCr/ATP negatively correlating with the basal PCr/ATP value (R = -0.74, P < 0.001). As NAA is a measure of mitochondrial function, there was also a significant negative correlation between basal NAA concentrations with the fractional change in PCr and ATP. Thus healthy human brain energetics is malleable and shifts with 7 days of Cr supplementation, with the regions of initially low PCr showing the largest increments in PCr. Overall, Cr supplementation appears to improve high-energy phosphate turnover in healthy brain and can result in either a decrease or an increase in high-energy phosphate concentrations.

31P MRS measurement of mitochondrial function in skeletal muscle: reliability, force-level sensitivity and relation to whole body maximal oxygen uptake

31P MRS measurement of mitochondrial function in skeletal muscle: reliability, force-level sensitivity and relation to whole body maximal oxygen uptake

Ions, transport, and energetics in normal and diseased skeletal muscle

Magnetic resonance spectroscopy (MRS) has highlighted the relationship between intracellular ionic homeostasis and the control of muscle energetics. In skeletal muscle, the oxidative rate of ATP synthesis is largely controlled by ADP, the concentration of which is determined by the creatine kinase equilibrium that includes the concentration of H+. At the onset of aerobic dynamic exercise, ATP is maintained largely by glycolysis, producing lactic acid and PCr breakdown. Vasodilation follows and ATP synthesis becomes predominantly oxidative. During recovery, PCr resynthesis gives a measure of mitochondrial function, and pH recovery reflects the Na+:H+ antiport activity. Dynamic31P MRS measurements can be used to derive quantitative information about the processes (fluxes and concentrations) described above. In diseased muscle (e.g., mitochondrial myopathy, dystrophy, hypertension) specific changes are observed in some of the control functions, ionic fluxes, or mitochondrial oxidative rates.

Metabolic abnormalities in skeletal muscle after myocardial infarction in the rat.

1. The effect of experimental myocardial infarction on exercise and recovery of rat skeletal muscle was studied using 31P n.m.r. 4 weeks post-operatively. 2. Myocardial infarction (12 +/- 3% of left ventricular volume), insufficient to produce haemodynamic manifestations of heart failure, was without significant effect on exercise bioenergetics of skeletal muscle. 3. Citrate synthase activity was reduced by 17% in the infarcted animals and there was a marked slowing of the rate of phosphocreatine recovery after infarction (half-time 0.7 +/- 0.1 min to 1.6 +/- 0.2 min) in the absence of evidence of left ventricular failure or hypertrophy. 4. The study of recovery bioenergetics could provide a more sensitive measure of mitochondrial function than exercise, where no bioenergetic abnormality was detected. 5. Myocardial infarction can produce evidence of mitochondrial abnormality in skeletal muscle in the absence of haemodynamic compromise.

Effects of UV irradiation on a living skin equivalent.

The Living Skin Equivalent (LSE) is an organotypic coculture composed of human dermal fibroblasts interspersed in a collagen-containing matrix and overlaid with human keratinocytes forming a stratified epidermis. The LSE has a dry, air-exposed epidermal surface suitable for the application of oils, creams and emulsions. These features suggested its feasibility as an in vitro skin model for studying the protective effects of sunscreens. Using the thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by various doses of UV-R (280-400 nm) or UV-A (320-400 nm) was evaluated in the LSE. The doses of UV radiation that caused 50% reductions in MTT conversion (UV-R50 or UV-A50) in different lots of LSE were 0.053 +/- 0.021 J/cm2 (n = 29) and 11.6 +/- 4.9 J/cm2 (n = 17) for UV-R and UV-A, respectively. The protective effects of an 8% homosylate standard and of five UV-A sunscreens, topically applied to the LSE, were determined and compared with their reported protection factors in human skin. Morphological changes and the release of proinflammatory mediators (interleukin-1-alpha, tumor necrosis factor-alpha and prostaglandin E2) implicated in UV-induced erythema were also demonstrated in the LSE exposed to UV-A or UV-B. The data suggest that the LSE can be used for studying the effects of UV radiation on skin and may have utility for assessing the efficacy of certain sunscreens against UV-B and UV-A.

The living skin equivalent as a model in vitro for ranking the toxic potential of dermal irritants

The living skin equivalent (LSE) is an organotypic co-culture composed of human dermal fibroblasts in a collagen-containing matrix overlaid with human keratinocytes that have formed a stratified epidermis. This model system was used as a dermatotoxicity model in vitro for studying the effects of test samples topically applied to the air-exposed epidermis. Using the colorimetric thiazolyl blue (MTT) conversion assay as a measure of mitochondrial function, the extent of cytotoxicity induced by several well-characterized chemical irritants was evaluated in the LSE. For the seven chemical irritants tested, the concentrations that inhibited MTT conversion by 50% were approximately those threshold concentrations at which irritation was seen in human skin. In addition, nine chemicals that were classified as non-irritating to human skin, including solids and water-insoluble substances, exhibited minimal or no inhibition of MTT conversion when tested at full strength. Time- and dose-dependent release of the pro-inflammatory mediators, prostaglandin E2, prostacyclin and interleukin-1-alpha (IL-1α) was seen in LSE exposed to two known chemical irritants, morpholine and hydroxylamine sulphate, but not in LSE exposed to a minimally irritating test sample. The permeability constants ( K p values) for water, caffeine, hydrocortisone, oestradiol and benzoic acid were determined in the LSE. In all cases the LSE was more permeable than human skin with K p values ranging from 0.56 ± 0.10 × 10 −3 cm/hr for hydrocortisone to 29 ± 5 × 10 −3 cm/hr for water penetration. The data suggest that organotypic skin cultures can be used as model systems for studying certain aspects of chemically induced dermal irritation. Using rates of water penetration as the most meaningful assessment of barrier competence, the LSE is approximately 30-fold more permeable than human skin. Although incomplete as a percutaneous absorption model, the presence of this partial barrier does influence the responses of cells in the LSE to topically applied chemicals.

Regulation of Mitochondrial Function and Biogenesis in Cucumber (Cucumis sativus L.) Cotyledons during Early Seedling Growth

The aim of this work was to characterize the respiratory metabolism of the greening cotyledons of cucumber (Cucumis sativus L.) during early seedling growth and to investigate how this is integrated with changes in mitochondrial biogenesis and function. In light-grown cotyledons, lipid mobilization extended from germination to 6 days postimbibition, reaching a maximum at 3 to 4 days postimbibition. The rate of dark oxygen uptake reached a maximum at 2 days postimbibition in dark-grown and 3 days postimbibition in light-grown cotyledons. Development of photosynthetic capacity occurred from 4 to 7 days postimbibition. In dark-grown cotyledons, lipid mobilization extended beyond 7 days postimbibition, and there was no greening or acquisition of photosynthetic competence. Measurements of mitochondrial function indicated that the respiratory capacity of the tissue changed such that during lipid mobilization there was a much greater capacity for the operation of the nondecarboxylating portion of the citric acid cycle (succinate to oxaloacetate), whereas during the development of photosynthetic function the activity of the remainder of the cycle (oxaloacetate to succinate) was induced. Comparison of the maximum capacities for mitochondrial substrate oxidations in vitro with the rates of in vivo substrate oxidations, predicted from the rate of lipid breakdown, indicated that mitochondria in this tissue operate at or below state 4 rates, suggesting limitation by both availability of ADP and substrate.

Changes in mitochondrial structure and function in 9l rat brain tumor cells treated in vitro with alpha-difluoromethylornithine, a polyamine biosynthesis inhibitor.

Mitochondrial structure and function were studied in 9L rat brain tumor cells depleted of polyamines by alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ornithine decarboxylase. Cells treated with methylglyoxal bis(guanylhydrazone), a reversible inhibitor of S-adenosylmethionine decarboxylase, were used for comparison because this polyamine biosynthesis inhibitor is known to cause structural and functional disruption of mitochondria. A significant increase in mitochondrial size, measured quantitatively, was found in alpha-difluoromethylornithine-treated cells (10 mM for 72 hr) compared with untreated cells (P < 0.001). This increase in mitochondrial size was reversed when putrescine was added to the cultures for 24 hr after alpha-difluoromethylornithine treatment. Putrescine alone had no effect on the size of mitochondria. Treatment of cells with methylglyoxal bis(guanylhydrazone) (80 muM for 48 hr) caused only a slight increase in mitochondrial size compared with mitochondria in untreated cells (P < 0.05) and failed to produce the dramatic ultrastructural changes reported in other cell lines. Ultrastructural examination revealed an increase in cytoplasmic and membrane-associated ribosomes in alpha-difluoromethylornithine-treated cells, an increase in cytoplasmic ribosomes in methylglyoxal bis(guanylhydrazone)-treated cells, and an increase in membrane-bound ribosomes in putrescine-treated cells. In cells treated first with alpha-difluoromethylornithine and then with putrescine, the distribution of ribosomes was normal. The distributions of ribosomes were not quantitatively assessed. Pyruvate utilization, a measure of mitochondrial function, was decreased in cells treated with 10 mM alpha-difluoromethylornithine for 72 hr, compared with untreated cells. Restoration of intracellular polyamine levels by the addition of putrescine 24 hr before analysis reversed this phenomenon. Putrescine treatment alone did not affect pyruvate utilization. Pyruvate utilization in methylglyoxal bis(guanylhydrazone)-treated cells was depressed to a greater extent than that in alpha-difluoromethylornithine-treated cells.