Activities Of Respiratory Chain Complexes Research Articles

Manganese Chloride Exposure Causes Disorder of Energy Metabolism and Induces Oxidative Stress and Autophagy in Chicken Liver.

Manganese (Mn) pollution is an important environmental problem because of the potential toxicity to human and animal health. However, the effects of Mn on energy metabolism and autophagy are not clear. Consequently, we examined the effects of excessive and chronic exposure to Mn on liver function, oxidative stress, respiratory chain complex activity, and autophagy in chicken liver. Our results indicated that the accumulation of Mn in the liver and levels of AST and ALT in the serum of the Mn-exposed group were significantly higher (P < 0.05) than those in the control group at 90 days; the activities of GSH-Px, SOD, CAT, Na+-K+-ATPase, Mg2+-ATPase, Ca2+-ATPase, and respiratory chain complexes (I, II, III) in the Mn-exposed group were significantly decreased (P < 0.05) compared to the control group. However, the MDA content, NO content, iNOS activity, mRNA and protein levels of iNOS, and autophagy-related genes in the Mn-exposed group were significantly increased (P < 0.05) compared to the control group. In contrast, the mRNA level and protein expression of mTOR were significantly decreased (P < 0.05) compared to the control group. Furthermore, the characteristic autophagic vacuolar organelles were observed in the Mn-exposed group. These results suggested that excess Mn exposure can cause a disorder of energy metabolism by mitochondrial injury and induce oxidative stress and autophagy, which eventually lead to liver damage.

Melatonin Inhibits Formation of Mitochondrial Permeability Transition Pores and Improves Oxidative Phosphorylation of Frozen-Thawed Ram Sperm.

Structural and functional damages to mitochondria of frozen-thawed sperm are a typical cryoinjury, with mitochondrial permeability transition pore (MPTP) formation being the hallmark change. Mitochondria are both a primary synthesis site and principle target for melatonin; this compound can directly inhibit MPTP formation and therefore confer protection at a mitochondrial level. The objective was to determine effects of melatonin on MPTP opening, viability, motility, and oxidative phosphorylation (OXPHOS) of frozen-thawed ram sperm. Ram semen was diluted in glucose-egg yolk buffer with 0 or 10−7 M melatonin (frozen and frozen + melatonin groups, respectively) and slow frozen, with fresh semen as Control. In frozen-thawed sperm, melatonin inhibited MPTP opening and lactate concentrations and improved sperm viability, motility, acetyl-CoA concentration and adenosine triphosphate (ATP) production. With regard to the underlying physiological mechanism, melatonin suppressed movement of citrate synthase, isocitrate dehydrogenase, oxoglutarate dehydrogenase complex, and F0F1-ATP synthase permeability from mitochondrial to cytosolic fractions induced by MPTP opening; furthermore, it increased mRNA expressions of respiratory chain complex components and activities of complexes I, II, III, and IV and thereby improved oxygen consumption capacity in frozen-thawed sperm. In conclusion, melatonin improved OXPHOS of frozen-thawed ram sperm, attributed to inhibition of cryopreservation-induced MPTP opening.

Skeletal muscle metabolism in sea-acclimatized king penguins. II. Improved efficiency of mitochondrial bioenergetics.

At fledging, juvenile king penguins (Aptenodytes patagonicus) must overcome the tremendous energetic constraints imposed by their marine habitat, including during sustained extensive swimming activity and deep dives in cold seawater. Both endurance swimming and skeletal muscle thermogenesis require high mitochondrial respiratory capacity while the submerged part of dive cycles repeatedly and greatly reduces oxygen availability, imposing a need for solutions to conserve oxygen. The aim of the present study was to determine in vitro whether skeletal muscle mitochondria become more 'thermogenic' to sustain heat production or more 'economical' to conserve oxygen in sea-acclimatized immature penguins (hereafter 'immatures') compared with terrestrial juveniles. Rates of mitochondrial oxidative phosphorylation were measured in permeabilized fibers and mitochondria from the pectoralis muscle. Mitochondrial ATP synthesis and coupling efficiency were measured in isolated muscle mitochondria. The mitochondrial activities of respiratory chain complexes and citrate synthase were also assessed. The results showed that respiration, ATP synthesis and respiratory chain complex activities in pectoralis muscles were increased by sea acclimatization. Furthermore, muscle mitochondria were on average 30-45% more energy efficient in sea-acclimatized immatures than in pre-fledging juveniles, depending on the respiratory substrate used (pyruvate, palmitoylcarnitine). Hence sea acclimatization favors the development of economical management of oxygen, decreasing the oxygen needed to produce a given amount of ATP. This mitochondrial phenotype may improve dive performance during the early marine life of king penguins, by extending their aerobic dive limit.

Astaxanthin attenuates d-galactose-induced brain aging in rats by ameliorating oxidative stress, mitochondrial dysfunction, and regulating metabolic markers.

Astaxanthin (AX) is a red-colored xanthophyll carotenoid with potent antioxidant, anti-inflammatory, and neuroprotective properties. However, the underlying in vivo mechanism by which AX protects the brain from oxidative stress remains unclear. In this study, we investigated the protective effect of AX on brain oxidative damage in a d-galactose-induced rat model of aging. We also explored its possible mechanism of action by analyzing the resulting serum metabolic profiles. Our results showed that AX significantly increased the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-Px) by 26%, 30%, and 53%, respectively. AX also significantly increased the mitochondrial membrane potential by 18% when compared with the model group. Additionally, treatment with AX (15 mg kg-1) increased the activities of respiratory chain complexes I and IV by 50.17% and 122.87%, respectively. Furthermore, AX also improved age-related morphological changes in the cerebral cortex and hippocampus. Significant differences in serum metabolic profiles were observed between the d-galactose and AX treatment groups. AX corrected amino acid metabolic problems by increasing the levels of N-acetyl-l-leucine, N-acetyl-l-tyrosine, and methionine sulfoxide to protect nerve cells. This also allowed AX to regulate the pentose phosphate pathway by acting on ergotoxine, d-xylose-5-phosphoric, and thiamine, to against oxidative stress and apoptosis. Moreover, AX reduced the levels of both hyodeoxycholic acid and chenodeoxycholic acid though the primary bile acid biosynthesis pathway, resulting in improved brain mitochondrial dysfunction. In conclusion, AX likely enhances the brain's antioxidant defenses through these potential metabolic means, enabling the brain to resist mitochondrial dysfunction, improve neuronal damage, and protect the electron transmission of mitochondrial respiratory chain, thus preventing brain aging.

Mitochondrial Respiratory Chain and Its Regulatory Elements SIRT1 and SIRT3 Play Important Role in the Initial Process of Energy Conversion after Moxibustion at Local Skin

Objectives To study how thermal energy is converted after moxibustion at local skin from the view of mitochondrial respiratory chain and its key regulatory elements of sirtuins 1 (SIRT1) and sirtuins 3 (SIRT3). Methods Two moxibustion temperatures usually used in clinical practice (38°C and 46°C) were applied to Zusanli (ST36) acupoint for 30 minutes in C57BL/6J mice. Local skin samples were harvested at 30 min and 72 h after moxibustion intervention, respectively. The activity of mitochondrial respiratory chain complexes I–V was detected by spectrophotometry. The expression of SIRT1 and SIRT3 protein was detected by immunofluorescence staining or western blot. Results Moxibustion at 38°C triggered more significant increase of mitochondrial respiratory chain complexes I–V expression. However, the protein expression of SIRT1 and SIRT3 at 46°C showed more obvious enhancement. In addition, the effect of mitochondrial respiratory chain complexes I–V activity on local skin of ST36 acupoint was more obvious at 30 min after moxibustion, while the expression of SIRT1 and SIRT3 protein was more significant at 72 h after moxibustion. Conclusion Mitochondrial respiratory chain and its key regulatory element proteins SIRT1 and SIRT3 play important role in the initial process of thermal energy conversion stimulated by different moxibustion temperatures in local skin.

Mitochondrial abundance and function in skeletal muscle and liver from Simmental beef cattle divergent for residual feed intake

Cellular mitochondrial function has been suggested to contribute to variation in feed efficiency (FE) among animals. The objective of this study was to determine mitochondrial abundance and activities of various mitochondrial respiratory chain complexes (complex I (CI) to complex IV (CIV)) in liver and muscle tissue from beef cattle phenotypically divergent for residual feed intake (RFI), a measure of FE. Individual DM intake (DMI) and growth were measured in purebred Simmental heifers (n = 24) and bulls (n = 28) with an initial mean BW (SD) of 372 kg (39.6) and 387 kg (50.6), respectively. All animals were offered concentrates ad libitum and 3 kg of grass silage daily, and feed intake was recorded for 70 days. Residuals of the regression of DMI on average daily gain (ADG), mid-test BW0.75 and backfat (BF), using all animals, were used to compute individual RFI coefficients. Animals were ranked within sex, by RFI into high (inefficient; top third of the population), medium (middle third of population) and low (efficient; bottom third of the population) terciles. Statistical analysis was carried out using the MIXED procedure of SAS v 9.3. Overall mean ADG (SD) and daily DMI (SD) for heifers were 1.2 (0.4) and 9.1 (0.5) kg, respectively, and for bulls were 1.8 (0.3) and 9.5 (1.02) kg, respectively. Heifers and bulls ranked as high RFI consumed 10% and 15% more (P < 0.05), respectively, than their low RFI counterparts. There was no effect of RFI on mitochondrial abundance in either liver or muscle (P > 0.05). An RFI × sex interaction was apparent for CI activity in muscle. High RFI animals had an increased activity (P < 0.05) of CIV in liver tissue compared to their low RFI counterparts; however, the relevance of that observation is not clear. Our data provide no clear evidence that cellular mitochondrial function within either skeletal muscle or hepatic tissue has an appreciable contributory role to overall variation in FE among beef cattle.

Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Mycelia Attenuates Doxorubicin-Induced Oxidative Stress and Upregulates Krebs Cycle Dehydrogenases Activity and ATP Level in Rat Brain.

Post-chemotherapy-induced cognitive dysfunction remains one of the challenges in cancer survivors. Cytokine-induced neurotoxicity manifests in subjects at any time after doxorubicin (DOX) chemotherapy. We examined the effect of bioactive Cordyceps militaris mycelia extract (CM) on the energy status, oxidative stress, and acetylcholinesterase activity in the brain of DOX treated rats. The CM (150 and 300 mg/kg b.w.) and DL-α lipoic acid (LA, 100 mg/kg b.w) were administered orally once daily for 5 days to male Wistar rats prior to the DOX administration (18 mg/kg as 3 doses of 6 mg/kg, i.p. b.w.) and continued for 6 more days. Cellular antioxidant status, Krebs cycle dehydrogenases, electron transport chain complexes (ETC) (I, III, and IV), adenosine triphosphate (ATP) level, advanced oxidation of protein products (AOPP), and acetylcholinesterase (AchE) activities were determined in the brain homogenate. The DOX alone treated group of animals showed significant decrease (p < 0.05) of brain antioxidant levels, Krebs cycle dehydrogenases activities, ETC complex activities, and decreased ATP level, while lipid peroxidation and AOPP levels were elevated. CM at 300 mg/kg b.w. or LA at 100 mg/kg b.w. elevated antioxidant status, Krebs cycle dehydrogenases, and complex activities and thus alleviated the toxicity. CM also inhibited the AchE activity in brain. The experimental results thus reveal that CM possessed excellent capacity to attenuate oxidative stress, upregulate respiratory chain complex activity and ATP levels, as well as inhibition of AchE activity.

Antimicrobial Susceptibility and Antibacterial Mechanism of Limonene against Listeria monocytogenes.

Limonene is a monoterpenoid compound, which is founded in a lot of plants’ essential oils with good antibacterial activity against food-borne pathogens, but it has an ambiguous antimicrobial susceptibility and mechanism against Listeria monocytogenes (L. monocytogenes). In this study, the antimicrobial susceptibility of Limonene to L. monocytogenes was studied, and some new sights regarding its antibacterial mechanism were further explored. Scanning electron microscopy (SEM) verified that limonene caused the destruction of the cell integrity and wall structure of L. monocytogenes. The increase in conductivity and the leakage of intracellular biomacromolecules (nucleic acids and proteins) confirmed that limonene had an obvious effect on cell membrane permeability. The results of Propidium Iodide (PI) fluorescence staining were consistent with the results of the conductivity measurements. This indicated that limonene treatment caused damage to the L. monocytogenes cell membrane. Furthermore, the decrease in ATP content, ATPase (Na+K+-ATPase, Ca2+-ATPase) activity and respiratory chain complex activity indicated that limonene could hinder ATP synthesis by inhibiting the activity of the respiratory complex and ATPase. Finally, differential expression of proteins in the respiratory chain confirmed that limonene affected respiration and energy metabolism by inhibiting the function of the respiratory chain complex.

Ginsenosides Rb1 and Rg1 Protect Primary Cultured Astrocytes against Oxygen-Glucose Deprivation/Reoxygenation-Induced Injury via Improving Mitochondrial Function.

This study aimed to evaluate whether ginsenosides Rb1 (20-S-protopanaxadiol aglycon) and Rg1 (20-S-protopanaxatriol aglycon) have mitochondrial protective effects against oxygen-glucose deprivation/reoxygenation (OGD/R)-induced injury in primary mouse astrocytes and to explore the mechanisms involved. The OGD/R model was used to mimic the pathological process of cerebral ischemia-reperfusion in vitro. Astrocytes were treated with normal conditions, OGD/R, OGD/R plus Rb1, or OGD/R plus Rg1. Cell viability was measured to evaluate the cytotoxicity of Rb1 and Rg1. Intracellular reactive oxygen species (ROS) and catalase (CAT) were detected to evaluate oxidative stress. The mitochondrial DNA (mtDNA) copy number and mitochondrial membrane potential (MMP) were measured to evaluate mitochondrial function. The activities of the mitochondrial respiratory chain (MRC) complexes I–V and the level of cellular adenosine triphosphate (ATP) were measured to evaluate oxidative phosphorylation (OXPHOS) levels. Cell viability was significantly decreased in the OGD/R group compared to the control group. Rb1 or Rg1 administration significantly increased cell viability. Moreover, OGD/R caused a significant increase in ROS formation and, subsequently, it decreased the activity of CAT and the mtDNA copy number. At the same time, treatment with OGD/R depolarized the MMP in the astrocytes. Rb1 or Rg1 administration reduced ROS production, increased CAT activity, elevated the mtDNA content, and attenuated the MMP depolarization. In addition, Rb1 or Rg1 administration increased the activities of complexes I, II, III, and V and elevated the level of ATP, compared to those in the OGD/R groups. Rb1 and Rg1 have different chemical structures, but exert similar protective effects against astrocyte damage induced by OGD/R. The mechanism may be related to improved efficiency of mitochondrial oxidative phosphorylation and the reduction in ROS production in cultured astrocytes.

HB-EGF Ameliorates Oxidative Stress-Mediated Uterine Decidualization Damage.

HB-EGF is essential for uterine decidualization, but its antioxidant function remains largely unclear. Here, we found that HB-EGF promoted the proliferation of stromal cells followed by the accelerated transition of the cell cycle from G1 to S phase and enhanced the expression or activity of Prl8a2, Prl3c1, and ALP which were well-established markers for uterine stromal cell differentiation during decidualization. Under oxidative stress, stromal cell differentiation was impaired, but this impairment was abrogated by rHB-EGF accompanied with the reduced levels of ROS and MDA which were regarded as the biomarkers for oxidative stress, indicating an antioxidant role of HB-EGF. Further analysis revealed that HB-EGF enhanced the activities of antioxidant enzymes SOD, CAT, and GPX, where addition of GPX inhibitor MS attenuated the induction of rHB-EGF on Prl8a2, Prl3c1, and ALP. Meanwhile, HB-EGF rescued the content of GSH and restored the ratio of GSH/GSSG after exposure to H2O2 but did not alter NOX activity. Along with a decline for mitochondrial superoxide, exogenous rHB-EGF improved the damage of oxidative stress on mtDNA copy number, ATP level, mitochondrial membrane potential, and activities of mitochondrial respiratory chain complex I and III whose blockage by ROT and AA led to a failure of rHB-EGF in protecting stromal cell differentiation against injury. Moreover, HB-EGF prevented stromal cell apoptosis by inhibiting Caspase-3 activity and Bax expression and recovering the level of Bcl-2 mRNA. Collectively, HB-EGF might ameliorate oxidative stress-mediated uterine decidualization damage.

Mouse Trmt2B protein is a dual specific mitochondrial metyltransferase responsible for m5U formation in both tRNA and rRNA

ABSTRACTRNA molecules of all species contain modified nucleotides and particularly m5U residues. The vertebrate mitochondrial small subunit rRNA contains m5U nucleotide in a unique site. In this work we found an enzyme, TRMT2B, responsible for the formation of this nucleotide and m5U residues in a number of mitochondrial tRNA species. Inactivation of the Trmt2B gene leads to a reduction of the activity of respiratory chain complexes I, III and IV, containing the subunits synthesized by the mitochondrial protein synthesis apparatus. Comparative sequence analysis of m5U-specific RNA methyltransferases revealed an unusual evolutionary pathway of TRMT2B formation which includes consecutive substrate specificity switches from the large subunit rRNA to tRNA and then to the small subunit rRNA.

Inhibition of receptor-interacting protein kinase 1 improves experimental non-alcoholic fatty liver disease

In non-alcoholic fatty liver disease (NAFLD), hepatocytes can undergo necroptosis: a regulated form of necrotic cell death mediated by the receptor-interacting protein kinase (RIPK) 1. Herein, we assessed the potential for RIPK1 and its downstream effector mixed lineage kinase domain-like protein (MLKL) to act as therapeutic targets and markers of activity in NAFLD. C57/BL6J-mice were fed a normal chow diet or a high-fat diet (HFD). The effect of RIPA-56, a highly specific inhibitor of RIPK1, was evaluated in HFD-fed mice and in primary human steatotic hepatocytes. RIPK1 and MLKL concentrations were measured in the serum of patients with NAFLD. When used as either a prophylactic or curative treatment for HFD-fed mice, RIPA-56 caused a downregulation of MLKL and a reduction of liver injury, inflammation and fibrosis, characteristic of non-alcoholic steatohepatitis (NASH), as well as of steatosis. This latter effect was reproduced by treating primary human steatotic hepatocytes with RIPA-56 or necrosulfonamide, a specific inhibitor of human MLKL, and by knockout (KO) of Mlklin fat-loaded AML-12 mouse hepatocytes. Mlkl-KO led to activation of mitochondrial respiration and an increase in β-oxidation in steatotic hepatocytes. Along with decreased MLKL activation, Ripk3-KO mice exhibited increased activities of the liver mitochondrial respiratory chain complexes in experimental NASH. In patients with NAFLD, serum concentrations of RIPK1 and MLKL increased in correlation with activity. The inhibition of RIPK1 improves NASH features in HFD-fed mice and reverses steatosis via an MLKL-dependent mechanism that, at least partly, involves an increase in mitochondrial respiration. RIPK1 and MLKL are potential serum markers of activity and promising therapeutic targets in NAFLD. There are currently no pharmacological treatment options for non-alcoholic fatty liver disease (NAFLD), which is now the most frequent liver disease. Necroptosis is a regulated process of cell death that can occur in hepatocytes during NAFLD. Herein, we show that RIPK1, a gatekeeper of the necroptosis pathway that is activated in NAFLD, can be inhibited by RIPA-56 to reduce not only liver injury, inflammation and fibrosis, but also steatosis in experimental models. These results highlight the potential of RIPK1 as a therapeutic target in NAFLD.

Mitochondrial Oxidative Stress Impairs Energy Metabolism and Reduces Stress Resistance and Longevity of C. elegans.

Introduction Mitochondria supply cellular energy and are key regulators of intrinsic cell death and consequently affect longevity. The nematode Caenorhabditis elegans is frequently used for lifespan assays. Using paraquat (PQ) as a generator of reactive oxygen species, we here describe its effects on the acceleration of aging and the associated dysfunctions at the level of mitochondria. Methods Nematodes were incubated with various concentrations of paraquat in a heat-stress resistance assay (37°C) using nucleic staining. The most effective concentration was validated under physiological conditions, and chemotaxis was assayed. Mitochondrial membrane potential (ΔΨm) was measured using rhodamine 123, and activity of respiratory chain complexes determined using a Clark-type electrode in isolated mitochondria. Energetic metabolites in the form of pyruvate, lactate, and ATP were determined using commercial kits. Mitochondrial integrity and structure was investigated using transmission electron microscopy. Live imaging after staining with fluorescent dyes was used to measure mitochondrial and cytosolic ROS. Expression of longevity- and mitogenesis-related genes were evaluated using qRT-PCR. Results PQ (5 mM) significantly increased ROS formation in nematodes and reduced the chemotaxis, the physiological lifespan, and the survival in assays for heat-stress resistance. The number of fragmented mitochondria significantly increased. The ∆Ψm, the activities of complexes I-IV of the mitochondrial respiratory chain, and the levels of pyruvate and lactate were significantly reduced, whereas ATP production was not affected. Transcript levels of genetic marker genes, atfs-1, atp-2, skn-1, and sir-2.1, were significantly upregulated after PQ incubation, which implicates a close connection between mitochondrial dysfunction and oxidative stress response. Expression levels of aak-2 and daf-16 were unchanged. Conclusion Using paraquat as a stressor, we here describe the association of oxidative stress, restricted energy metabolism, and reduced stress resistance and longevity in the nematode Caenorhabditis elegans making it a readily accessible in vivo model for mitochondrial dysfunction.

Activity of respiratory chain complexes of peripheral blood mononuclear cells as marker of mitochondrial dysfunction in acute cardiovascular pathology

Mitochondrial respiratory chain enzyme activities were determined by spectrophotometric enzyme assay in lymphocytes of 100 patients with acute myocardial infarction. A significant decrease in the activity of complexes II–III and IV and an increase in the activity of complex I of the respiratory chain were found. According to the correlation analysis, the feedback of troponin T and I concentrations in the serum and the activity of complex IV of the respiratory chain was detected. A decrease in the activity of the IV complex of the mitochondrial lymphocyte respiratory chain below 2.87 IU/g of protein indicates an increased level of clinical risk in patients with acute myocardial infarction.

Toxicity of thifluzamide in earthworm (Eisenia fetida)

An increase in the area treated with the fungicide thifluzamide has triggered concerns for soil ecosystem service providers such as earthworms. Here, we assessed effects of thifluzamide on earthworm (Eisenia fetida) biomarker indicators of stress responses and reproduction following exposure to 0, 0.1, 1.0, and 10.0 mg of thifluzamide kg−1 soil for 7, 14, 21, and 28 d (biomarker indicators) and 30 d (reproduction). Growth and reproduction were inhibited by exposure to thifluzamide at 10.0 mg/kg, and the activities of succinate dehydrogenase (SDH) and respiratory chain complex II were inhibited by exposure to 1.0 and 10.0 mg/kg thifluzamide for the majority of the 28-d experiment. Reactive oxygen species (ROS) increased across all thifluzamide treatments, and the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) tended to be inhibited by thifluzamide. Upon exposure to thifluzamide, the activities of catalase (CAT) and guaiacol peroxidase (POD) initially increased and then decreased. Increased levels of malondialdehyde (MDA) were detected only at seven days after exposure, and genotoxicity increased as the thifluzamide concentration increased. The results suggest that thifluzamide presents a potential risk to earthworms at the concentration of 10.0 mg/kg, and its use should be moderated to reduce damage to soil ecosystem function.

Aberrant mitochondrial bioenergetics in the cerebral cortex of the Fmr1 knockout mouse model of fragile X syndrome.

Impaired energy metabolism may play a role in the pathogenesis of neurodevelopmental disorders including fragile X syndrome (FXS). We checked brain energy status and some aspects of cell bioenergetics, namely the activity of key glycolytic enzymes, glycerol-3-phosphate shuttle and mitochondrial respiratory chain (MRC) complexes, in the cerebral cortex of the Fmr1 knockout (KO) mouse model of FXS. We found that, despite a hyperactivation of MRC complexes, adenosine triphosphate (ATP) production via mitochondrial oxidative phosphorylation (OXPHOS) is compromised, resulting in brain energy impairment in juvenile and late-adult Fmr1 KO mice. Thus, an altered mitochondrial energy metabolism may contribute to neurological impairment in FXS.

Hepatotoxicity and mechanism study of chrysophanol-8-O-glucoside in vitro

To better understand the hepatotoxicity of anthraquinone glycosides, the hepatotoxicity of six anthraquinone glycosides was evaluated. The results show that chrysophanol-8-O-glucoside(C8G) has strong hepatotoxicity and can lead to increased LDH leakage and ROS, decreased GSH and MMP in L-02 hepatocytes. The results of C8G hepatotoxicity proteomics shows that, a total of 773 differentially expressed proteins were screened and analyzed using GO analysis and Pathway enrichment analysis. Our results show that C8G can lead to abnormal oxidative phosphorylation by inhibiting the function of mitochondrial complexes, resulting in decreased mitochondrial membrane potential (MMP), increased reactive oxygen species (ROS), and eventually resulting in mitochondrial damage and apoptosis. Western blot results verified the accuracy of quantitative proteomic results, and also evaluated the expression of Bax, caspase-3, -8, -9, Bcl-2, Cyt C in the mitochondria and cytosolic. The mitochondrial respiratory chain complexes activity assay result also confirmed that C8G could inhibit the activity of all mitochondrial complexes. The results of this study indicate that the hepatotoxicity mechanism of C8G is related to mitochondrial dysfunction, especially the mitochondrial complex function.

Novel role of Tieg1 in muscle metabolism and mitochondrial oxidative capacities.

Tieg1 is involved in multiple signalling pathways, human diseases, and is highly expressed in muscle where its functions are poorly understood. We have utilized Tieg1 knockout (KO) mice to identify novel and important roles for this transcription factor in regulating muscle ultrastructure, metabolism and mitochondrial functions in the soleus and extensor digitorum longus (EDL) muscles. RNA sequencing, immunoblotting, transmission electron microscopy, MRI, NMR, histochemical and mitochondrial function assays were performed. Loss of Tieg1 expression resulted in altered sarcomere organization and a significant decrease in mitochondrial number. Histochemical analyses demonstrated an absence of succinate dehydrogenase staining and a decrease in cytochrome c oxidase (COX) enzyme activity in KO soleus with similar, but diminished, effects in the EDL. Decreased complex I, COX and citrate synthase (CS) activities were detected in the soleus muscle of KO mice indicating altered mitochondrial function. Complex I activity was also diminished in KO EDL. Significant decreases in CS and respiratory chain complex activities were identified in KO soleus. 1 H-NMR spectra revealed no significant metabolic difference between wild-type and KO muscles. However, 31 P spectra revealed a significant decrease in phosphocreatine and ATPγ. Altered expression of 279 genes, many of which play roles in mitochondrial and muscle function, were identified in KO soleus muscle. Ultimately, all of these changes resulted in an exercise intolerance phenotype in Tieg1 KO mice. Our findings have implicated novel roles for Tieg1 in muscle including regulation of gene expression, metabolic activity and organization of tissue ultrastructure. This muscle phenotype resembles diseases associated with exercise intolerance and myopathies of unknown consequence.

Mitochondrial Function in Peripheral Blood Mononuclear Cells (PBMC) Is Enhanced, Together with Increased Reactive Oxygen Species, in Severe Asthmatic Patients in Exacerbation.

Asthma is a chronic inflammatory lung syndrome with an increasing prevalence and a rare but significant risk of death. Its pathophysiology is complex, and therefore we investigated at the systemic level a potential implication of oxidative stress and of peripheral blood mononuclear cells’ (PBMC) mitochondrial function. Twenty severe asthmatic patients with severe exacerbation (GINA 4–5) and 20 healthy volunteers participated at the study. Mitochondrial respiratory chain complexes activities using different substrates and reactive oxygen species (ROS) production were determined in both groups by high-resolution respirometry and electronic paramagnetic resonance, respectively. Healthy PBMC were also incubated with a pool of plasma of severe asthmatics or healthy controls. Mitochondrial respiratory chain complexes activity (+52.45%, p = 0.015 for VADP) and ROS production (+34.3%, p = 0.02) were increased in asthmatic patients. Increased ROS did not originate mainly from mitochondria. Plasma of severe asthmatics significantly increased healthy PBMC mitochondrial dioxygen consumption (+56.8%, p = 0.031). In conclusion, such asthma endotype, characterized by increased PMBCs mitochondrial oxidative capacity and ROS production likely related to a plasma constituent, may reflect activation of the immune system. Further studies are needed to determine whether increased PBMC mitochondrial respiration might have protective effects, opening thus new therapeutic approaches.

P.79Malignant cardiac phenotype after pressure overload in autosomal-dominant desminopathies: Lessons from heterozygous R349P desmin knock-in mice

Which etiological factors significantly aggravate the disease progression in genetically determined forms of cardiomyopathies? In the present study, we investigated the influence of pressure overload on cardiac function in our R349P desmin knock-in mouse model for human desminopathies. We performed transverse aortic constriction (TAC) operations in heterozygous R349P desmin knock-in mice and wild-type siblings. Left heart catheterization demonstrated effective TAC procedures by marked increases in systolic carotid pressure and end-systolic and end-diastolic left ventricular pressure values. Echocardiography demonstrated a significant reduction of the left ventricular ejection fraction only in TAC R349P desmin knock-in mice as compared to controls. Electrophysiological investigation revealed an increase in spontaneous AV blocks and the longest AV block duration in the mutant TAC mice. Furthermore, the probability of induction of ventricular tachycardias was also significantly increased in these mice. Mitochondrial analyses demonstrated marked reductions of the activities of respiratory chain complexes I and IV as well as significantly reduced mtDNA copy numbers in the TAC-operated desminopathy mice. While young, 3-month-old, non-operated heterozygous R349P desmin knock-in mice do not show obvious signs of cardiomyopathy, they develop marked signs of cardiomyopathy with accompanying arrhythmias and AV blocks when subjected to pressure overload by TAC operation. The cardiac disease manifestations are associated with reductions in respiratory chain complex activities and mtDNA copy numbers. Preventive avoidance of pressure overload, i.e. arterial hypertension, to the heart might represent a crucial therapeutic measure.