BACKGROUND: Mitochondrial diseases belong to the most severe inherited metabolic disorders affecting pediatric population. Despite detailed knowledge of mtDNA mutations and progress in identification of affected nuclear genes, diagnostics of a substantial part of mitochondrial diseases relies on clinical symptoms and biochemical data from muscle biopsies and cultured fibroblasts. METHODS: To investigate manifestation of oxidative phosphorylation defects in isolated lymphocytes, digitonin-permeabilized cells from 48 children were analyzed by high resolution respirometry, cytofluorometric detection of mitochondrial membrane potential and immunodetection of respiratory chain proteins with SDS and Blue Native electrophoreses. RESULTS: Evaluation of individual respiratory complex activities, ATP synthesis, kinetic parameters of mitochondrial respiratory chain and the content and subunit composition of respiratory chain complexes enabled detection of inborn defects of respiratory complexes I, IV and V within 2 days. Low respiration with NADH-dependent substrates and increased respiration with glycerol-3-phosphate revealed complex I defects; changes in p 50 for oxygen and elevated uncoupling control ratio pointed to complex IV deficiency due to SURF1 or SCO2 mutation; high oligomycin sensitivity of state 3-ADP respiration, upregulated mitochondrial membrane potential and low content of complex V were found in lymphocytes with ATP synthase deficiency due to TMEM70 mutations. CONCLUSION: Based on our results, we propose the best biochemical parameters predictive for defects of respiratory complexes I, IV and V manifesting in peripheral blood lymphocytes. GENERAL SIGNIFICANCE: The noninvasiveness, reliability and speed of an approach utilizing novel biochemical criteria demonstrate the high potential of isolated lymphocytes for diagnostics of oxidative phosphorylation disorders in pediatric patients.

• Publikační typ
• časopisecké články MeSH

Impaired myocardial bioenergetics is a hallmark of many cardiac diseases. There is a need of a simple and reproducible method of assessment of mitochondrial function from small human myocardial tissue samples. In this study we adopted high-resolution respirometry to homogenates of fresh human cardiac muscle and compare it with isolated mitochondria. We used atria resected during cardiac surgery (n = 18) and atria and left ventricles from brain-dead organ donors (n = 12). The protocol we developed consisting of two-step homogenization and exposure of 2.5% homogenate in a respirometer to sequential addition of 2.5 mM malate, 15 mM glutamate, 2.5 mM ADP, 10 μM cytochrome c, 10 mM succinate, 2.5 μM oligomycin, 1.5 μM FCCP, 3.5 μM rotenone, 4 μM antimycin and 1 mM KCN or 100 mM Sodium Azide. We found a linear dependency of oxygen consumption on oxygen concentration. This technique requires < 20 mg of myocardium and the preparation of the sample takes <20 min. Mitochondria in the homogenate, as compared to subsarcolemmal and interfibrillar isolated mitochondria, have comparable or better preserved integrity of outer mitochondrial membrane (increase of respiration after addition of cytochrome c is up to 11.7±1.8% vs. 15.7±3.1%, p˂0.05 and 11.7±3.5%, p = 0.99, resp.) and better efficiency of oxidative phosphorylation (Respiratory Control Ratio = 3.65±0.5 vs. 3.04±0.27, p˂0.01 and 2.65±0.17, p˂0.0001, resp.). Results are reproducible with coefficient of variation between two duplicate measurements ≤8% for all indices. We found that whereas atrial myocardium contains less mitochondria than the ventricle, atrial bioenergetic profiles are comparable to left ventricle. In conclusion, high resolution respirometry has been adapted to homogenates of human cardiac muscle and shown to be reliable and reproducible.

• MeSH
• citrátsynthasa metabolismus   MeSH
• dospělí MeSH
• energetický metabolismus MeSH
• kryoprezervace MeSH
• kyslík metabolismus   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mastné kyseliny metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• oxidace-redukce MeSH
• senioři MeSH
• srdeční mitochondrie metabolismus   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Using skeletal muscle homogenates for respirometry has many advantages, but the main challenge is avoiding the damage to outer mitochondrial membrane (OMM) and complex I. By optimising the amount of muscle and careful titration of substrates and inhibitors we developed a new protocol and compared it to isolated mitochondria. We found acceptable damage to OMM (~10-15% increment of oxygen flux after addition of cytochrome c) and to complex I (~70% of electron flux). Homogenate retained ~90% of phosphorylation capacity of isolated mitochondria. The use of fresh homogenate was crucial as mitochondrial function declined rapidly after 2-3h of cold storage.

• MeSH
• buněčné dýchání * MeSH
• buněčné extrakty MeSH
• cytologické techniky metody   MeSH
• dospělí MeSH
• jehlová biopsie MeSH
• kosterní svaly fyziologie   MeSH
• lidé středního věku MeSH
• lidé MeSH
• mitochondriální nemoci diagnóza   MeSH
• mitochondrie fyziologie   MeSH
• odběr biologického vzorku metody   MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• senioři nad 80 let MeSH
• senioři MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Studie se zaměřuje na hodnocení účinku opakované expozice HBO na mitochondriální respiraci lidských fetálních plicních fibroblastů (HFL1). Mitochondriální spotřeba kyslíku byla hodnocena metodou high-resolution respirometry (HRR), životaschopnost buněk hodnocena PrestoBlue®, morfologie analyzovaná rutinní fluorescenční mikroskopií a fázovým kontrastem a byla zhodnocena aktivita superoxid dismutázy (SOD) a citrát syntázy (CS). Buňky byly vystaveny HBO (3ATA) 2 hodiny denně po dobu 5 po sobě jdoucích dní. Den po poslední HBO expozici vykazovaly buňky podstatně menší plochu a obvod, sníženou životaschopnost a zvýšenou aktivitu SOD. Nebyly zjištěny žádné změny v aktivitě CS ani v kvalitě mitochondriální sítě. HRR odhalila sníženou mitochondriální spotřebu kyslíku, která se projevila snížením aktivity komplexu II a sníženou spotřebou kyslíku při oxidací mastných kyselin. Naše zjištění dokládají, že v podmínkách napodobujících intermitentní expozici HBO, trpí plicní fibroblasty zhoršenou mitochondriální respirací spojenou s komplexem II a poruchou buněčného růstu i při zvýšené antioxidační obraně. Mechanismus této HBO-indukované mitochondriální dysfunkce je nutné hlouběji prozkoumat.

Study aims to evaluate effects of repeated exposure to HBO on mitochondrial respiration assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities using human lung fibroblasts. The cells were exposed to HBO (3 ATA) for 2 hours per day for 5 consecutive days. One day after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.

• Klíčová slova
• respirometrie,
• MeSH
• buněčné dýchání MeSH
• fibroblasty * enzymologie   metabolismus   MeSH
• fluorescenční mikroskopie MeSH
• hyperbarická oxygenace * škodlivé účinky   MeSH
• hyperoxie MeSH
• lidé MeSH
• mitochondrie MeSH
• plíce anatomie a histologie   MeSH
• plod MeSH
• superoxiddismutasa MeSH
• techniky in vitro MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• práce podpořená grantem MeSH

Platelet mitochondria can be used in the study of mitochondrial dysfunction in various complex diseases and can help in finding biological markers for diagnosing the disease, monitoring its course and the effects of treatment. The aim of this chapter was to describe in detail the method of measuring mitochondrial respiration in platelets using high-resolution respirometry. The described method was successfully used for the study of mitochondrial dysfunction in neuropsychiatric diseases.

• MeSH
• buněčné dýchání MeSH
• lidé MeSH
• mitochondrie metabolismus   MeSH
• polarografie přístrojové vybavení   metody   MeSH
• trombocyty metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Oxidative phosphorylation is a key process of intracellular energy transfer by which mitochondria produce ATP. Isolated mitochondria serve as a biological model for understanding the mitochondrial respiration control, effects of various biologically active substances, and pathophysiology of mitochondrial diseases. The aim of our study was to evaluate pig brain mitochondria as a proper biological model for investigation of activity of the mitochondrial electron transport chain. Oxygen consumption rates of isolated pig brain mitochondria were measured using high-resolution respirometry. Mitochondrial respiration of crude mitochondrial fraction, mitochondria purified in sucrose gradient, and mitochondria purified in Percoll gradient were assayed as a function of storage time. Oxygen flux and various mitochondrial respiratory control ratios were not changed within two days of mitochondria storage on ice. Leak respiration was found higher and Complex I-linked respiration lower in purified mitochondria compared to the crude mitochondrial fraction. Damage to both outer and inner mitochondrial membrane caused by the isolation procedure was the greatest after purification in a sucrose gradient. We confirmed that pig brain mitochondria can serve as a biological model for investigation of mitochondrial respiration. The advantage of this biological model is the stability of respiratory parameters for more than 48 h and the possibility to isolate large amounts of mitochondria from specific brain areas without the need to kill laboratory animals. We suggest the use of high-resolution respirometry of pig brain mitochondria for research of the neuroprotective effects and/or mitochondrial toxicity of new medical drugs.

• MeSH
• biologické modely * MeSH
• buněčné dýchání MeSH
• citrátsynthasa metabolismus   MeSH
• kyslík metabolismus   MeSH
• mitochondriální membrány metabolismus   MeSH
• mitochondrie metabolismus   MeSH
• mozek metabolismus   MeSH
• Sus scrofa MeSH
• transport elektronů MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Measurement of oxygen consumption of cultured cells is widely used for diagnosis of mitochondrial diseases, drug testing, biotechnology, and toxicology. Fibroblasts are cultured in monolayers, but physiological measurements are carried out in suspended or attached cells. We address the question whether respiration differs in attached versus suspended cells using multiwell respirometry (Agilent Seahorse XF24) and high-resolution respirometry (Oroboros O2k), respectively. Respiration of human dermal fibroblasts measured in culture medium was baseline-corrected for residual oxygen consumption and expressed as oxygen flow per cell. No differences were observed between attached and suspended cells in ROUTINE respiration of living cells and LEAK respiration obtained after inhibition of ATP synthase by oligomycin. The electron transfer capacity was higher in the O2k than in the XF24. This could be explained by a limitation to two uncoupler titrations in the XF24 which led to an underestimation compared to multiple titration steps in the O2k. A quantitative evaluation of respiration measured via different platforms revealed that short-term suspension of fibroblasts did not affect respiratory activity and coupling control. Evaluation of results obtained by different platforms provides a test for reproducibility beyond repeatability. Repeatability and reproducibility are required for building a validated respirometric database.

• MeSH
• buněčné dýchání * fyziologie   MeSH
• fibroblasty MeSH
• lidé MeSH
• oxidativní fosforylace * MeSH
• reprodukovatelnost výsledků MeSH
• spotřeba kyslíku fyziologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

CONTEXT: Monoestolides belonging to the fatty acid-hydroxy fatty acid (FAHFA) family have recently emerged as promising insulin sensitizers. OBJECTIVE: To investigate and compare impact of two selected FAHFA isomers, namely 9-hexadecanoyloxy-octadecanoic acid [9-PAHSA] and 9-(9Z-octadecenoyloxy)-octadecanoic acid [9-OAHSA], on intact livers in C57BL/6J mice. MATERIALS AND METHODS: Short-term in vivo study with intragastric gavage of 13 mg/kg of substances. Morphological, biochemical and high-resolution respirometric assessment of plasma and liver tissue or homogenates thereof. RESULTS: The 9-OAHSA-gavaged mice had the highest final total body weight, the lowest free fatty acid circulating levels and the highest plasma activities of both ALT and AST. No significant changes of ambient glycaemia were found, however 9-PAHSA-gavaged mice tended to have lower glycaemia than other animals. Respirometry proved no substance-dependent differences. DISCUSSION AND CONCLUSION: 9-PAHSA was more metabolically beneficial and less hepatotoxic than 9-OAHSA. Bioenergetic machinery of liver homogenates seemed unaffected at our FAHFA dose.

• MeSH
• inzulin * MeSH
• játra MeSH
• mastné kyseliny * MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) are widely used drugs for lowering blood lipid levels and preventing cardiovascular diseases. However, statins can have serious adverse effects, which may be related to development of mitochondrial dysfunctions. The aim of study was to demonstrate the in vivo effect of high and therapeutic doses of statins on mitochondrial respiration in blood platelets. Model approach was used in the study. Simvastatin was administered to rats at a high dose for 4 weeks. Humans were treated with therapeutic doses of rosuvastatin or atorvastatin for 6 weeks. Platelet mitochondrial respiration was measured using high-resolution respirometry. In rats, a significantly lower physiological respiratory rate was found in intact platelets of simvastatin-treated rats compared to controls. In humans, no significant changes in mitochondrial respiration were detected in intact platelets; however, decreased complex I-linked respiration was observed after statin treatment in permeabilized platelets. We propose that the small in vivo effect of statins on platelet energy metabolism can be attributed to drug effects on complex I of the electron transport system. Both intact and permeabilized platelets can be used as a readily available biological model to study changes in cellular energy metabolism in patients treated with statins.

• MeSH
• buněčné dýchání účinky léků   MeSH
• dyslipidemie farmakoterapie   MeSH
• krysa rodu rattus MeSH
• lidé MeSH
• mitochondrie účinky léků   MeSH
• statiny škodlivé účinky   MeSH
• trombocyty účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu rattus MeSH
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Mitochondria represent a possible drug target with unexplored therapeutic and toxicological potential. The possibility was suggested that antidepressants, mood stabilizers and other drugs may show some therapeutic and/or toxic effects through their action on mitochondrial functions. There are no sufficient data about the effect of these drugs on mitochondrial respiration in the brain. We investigated the in vitro effects of amitriptyline, fluoxetine, tianeptine, ketamine, lithium, valproate, olanzapine, chlorpromazine and propranolol on mitochondrial respiration in crude mitochondrial fractions of pig brains. Respiration was energized using substrates of complex I or complex II and dose dependent drug-induced changes in mitochondrial respiratory rate were measured by high-resolution respirometry. Antidepressants, but not mood stabilizers, ketamine and propranolol were found to inhibit mitochondrial respiratory rate. The effective dose of antidepressants reaching half the maximal respiratory rate was in the range of 0.07-0.46 mmol/L. Partial inhibition was found for all inhibitors. Differences between individual drugs with similar physicochemical properties indicate selectivity of drug-induced changes in mitochondrial respiratory rate. Our findings suggest that mood stabilizers do not interfere with brain mitochondrial respiration, whereas direct mitochondrial targeting is involved in mechanisms of action of pharmacologically different antidepressants.

• MeSH
• antidepresiva toxicita   MeSH
• buněčné dýchání účinky léků   MeSH
• down regulace MeSH
• energetický metabolismus účinky léků   MeSH
• mitochondrie účinky léků   metabolismus   MeSH
• mozková kůra účinky léků   metabolismus   MeSH
• prasata MeSH
• trankvilizéry toxicita   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH