Levels Of Mitochondrial Respiratory Complexes Research Articles

Vinpocetine and coenzyme Q10 combination alleviates cognitive impairment caused by ionizing radiation by improving mitophagy

ObjectiveThis research was designed to ascertain the effect and mechanism of vinpocetine (VIN) and coenzyme Q10 (CoQ10) combination on cognitive impairment induced by ionizing radiation (IR). MethodsCognitive impairment in mice was induced by 9-Gy IR, and they were intraperitoneally injected with VIN, CoQ10, or VIN + CoQ10. Then novel object recognition and Morris water maze tests were used to detect cognitive function. The number of hippocampal neurons and BrdU+Dcx+ cells was observed by Nissl and immunofluorescence staining. Mitochondrial respiratory complex I, adenosine triphosphate (ATP), and mitochondrial membrane potential (MMP) were evaluated, as well as oxidative stress injury. Mitophagy in hippocampal neurons was evaluated by observing the ultrastructure of hippocampal neurons and assessing the expression of mitophagy-related proteins. ResultsIR reduced novel object discrimination index, the time for platform crossing, and the time spent in platform quadrant, in addition to neuron loss, downregulated levels of mitochondrial respiratory complex I, ATP, and MMP, aggravated oxidative stress injury, increased expression of LC3 II/I, Beclin1, PINK1, and parkin, and decreased P62 expression. VIN or CoQ10 treatment mitigated cognitive dysfunction, neurons loss, mitochondrial damage, and oxidative stress injury, and enhanced mitophagy in hippocampal neurons. VIN and CoQ10 combination further protected against IR-induced cognitive dysfunction than VIN or CoQ10 alone. ConclusionVIN combined with CoQ10 improved neuron damage, promoted mitophagy, and ameliorated cognitive impairment in IR mice.

Establishment of a platform for measuring mitochondrial oxygen consumption rate for cardiac mitochondrial toxicity.

The heart has an abundance of mitochondria since cardiac muscles require copious amounts of energy for providing continuous blood through the circulatory system, thereby implying that myocardial function is largely reliant on mitochondrial energy. Thus, cardiomyocytes are susceptible to mitochondrial dysfunction and are likely targets of mitochondrial toxic drugs. Various methods have been developed to evaluate mitochondrial toxicity by evaluating toxicological mechanisms, but an optimized and standardized assay for cardiomyocytes remains unmet. We have therefore attempted to standardize the evaluation system for determining cardiac mitochondrial toxicity, using AC16 human and H9C2 rat cardiomyocytes. Three clinically administered drugs (acetaminophen, amiodarone, and valproic acid) and two anticancer drugs (doxorubicin and tamoxifen) which are reported to have mitochondrial effects, were applied in this study. The oxygen consumption rate (OCR), which directly reflects mitochondrial function, and changes in mRNA levels of mitochondrial respiratory complex I to complex V, were analyzed. Our results reveal that exposure to all five drugs results in a concentration-dependent decrease in the basal and maximal levels of OCR in AC16 cells and H9C2 cells. In particular, marked reduction in the OCR was observed after treatment with doxorubicin. The reduction in OCR after exposure to mitochondrial toxic drugs was found to be associated with reduced mRNA expression in the mitochondrial respiratory complexes, suggesting that the cardiac mitochondrial toxicity of drugs is majorly due to dysfunction of mitochondrial respiration. Based on the results of this study, we established and standardized a protocol to measure OCR in cardiomyocytes. We expect that this standardized evaluation system for mitochondrial toxicity can be applied as basic data for establishing a screening platform to evaluate cardiac mitochondrial toxicity of drugs, during the developmental stage of new drug discovery.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5–10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

Abstract 285: Osteopontin Deficiency Ameliorates Heart Failure with Preserved Ejection Fraction <HFpEF> Pathology by Upregulating Mitochondrial 2-Oxoglutarate Dehydrogenase Like <OGDHL> Enzyme

HFpEF is an increasingly prevalent syndrome associated with impaired myocardial energetics, for which no etiologic therapy is available. Osteopontin (OPN) is a matricellular protein that is upregulated in the circulation of HFpEF patients, and reported to induce mitochondrial stress in rodent cardiomyocytes. Here we evaluate the role of circulating OPN in regulating myocardial function in the nephrotic Col4a3 -/- mouse model of HFpEF. We performed extensive cardiac, biochemical and mitochondrial analyses of the Col4a3 -/- mouse and found a striking HFpEF phenotype. We showed OPN levels were elevated in Col4a3 -/- mice (FC=2.1, n=6; p<.01). Col4a3 -/- mice were hypertensive, had diastolic dysfunction, myocyte hypertrophy and interstitial fibrosis - all of which were ameliorated in Col4a3 -/- OPN -/- mice (n=5-20; p<.05). Col4a3 -/- hearts had dysmorphic mitochondria (EM), lowered antioxidant capacity as a 50% reduction in GSH/GSSG ratio (n=6; p<.05) and lower protein levels of mitochondrial respiratory complexes I, II and IV (p<.05). Flux assay in adult cardiomyocytes showed that maximal respiration was reduced in Col4a3 -/- hearts (575.84±37.6 vs 322.34±25.48 pmol/min in WT, n=9; p<.0001). Microarray data (validated by mitochondrial blot) implicated OGDHL as decreased in Col4a3 -/- hearts but increased in double knockout Col4a3 -/- OPN -/- hearts compared to WT (n=3; p<.05). OGDH activity was also lower in Col4a3 -/- hearts (17.1±7.3 vs 2.5±1.1 mU/mg in WT; n=6; p<.05). In Col4a3 -/- mice, heart-specific AAV9-mediated overexpression of OGDHL, similar to global OPN KO, improved survival by ~50-100% (p<.0001). Isovolumetric relaxation time, a marker of diastolic dysfunction, which is prolonged in Col4a3 -/- mice (26.17 vs 15.30±1 ms, n=26; p<.001) was decreased in Col4a3 -/- OPN -/- mice (18.1±1 ms, n=37; p<.01) as well as in AAV9-cTnT-OGDHL-treated Col4a3 -/- mice (16.7±2.5 ms, n=8; p<.05). In conclusion, we present a new mouse model for HFpEF in which diastolic function and lifespan can be improved by genetic deletion of OPN or cardiac OGDHL gene therapy. Our results elucidate for the first time the pivotal roles of circulating OPN and cardiac OGDHL in HFpEF pathophysiology and present two related potential therapeutic targets for HFpEF.

Mitochondrial function contributes to oxysterol-induced osteogenic differentiation in mouse embryonic stem cells

Oxysterols, oxidized derivatives of cholesterol, are biologically active molecules. Specific oxysterols have potent osteogenic properties that act on osteoprogenitor cells. However, the molecular mechanisms underlying these osteoinductive effects on embryonic stem cells (ESCs) are unknown. This study investigated the effect of an oxysterol combination of 22(S)-hydroxycholesterol and 20(S)-hydroxycholesterol (SS) on osteogenic differentiation of ESCs and the alterations to mitochondrial activity during differentiation. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, matrix mineralization, mRNA expression of osteogenic factors, runt-related transcription factor 2, osterix, and osteocalcin, and protein levels of collagen type IA (COLIA) and osteopontin (OPN). Treatment of cells with SS increased osteoinductive activity compared to the control group. Intracellular reactive oxygen species production, intracellular ATP content, mitochondrial membrane potential, mitochondrial mass, mitochondrial DNA copy number, and mRNA expression of peroxisome proliferator-activated receptor-γ coactivators 1α and β, transcription factors involved in mitochondrial biogenesis, were significantly increased during osteogenesis, indicating upregulation of mitochondrial activity. Oxysterol combinations also increased protein levels of mitochondrial respiratory complexes I–V. We also found that SS treatment increased hedgehog signaling target genes, Smo and Gli1 expression. Inhibition of Hh signaling by cyclopamine suppressed mitochondrial biogenesis and ESC osteogenesis. Subsequently, oxysterol-induced Wnt/β-catenin pathways were inhibited by repression of Hh signaling and mitochondrial biogenesis. Transfection of β-catenin specific siRNA decreased the protein levels of COLIA and OPN, as well as ALP activity. Collectively, these data suggest that lipid-based oxysterols enhance differentiation of ESCs toward the osteogenic lineage by regulating mitochondrial activity, canonical Hh/Gli, and Wnt/β-catenin signaling.

S54 Preservation of mitochondrial oxidative capacity in critically ill patients balances reduction in mitochondrial biogenesis

Background and Aims In severe sepsis, early mitochondrial dysfunction in skeletal muscle is associated with decreased biogenesis and adverse patient outcome. We hypothesised that reduction in mitochondrial content during critical illness would be balanced by enhanced capacity to produce ATP achieved by switching from glucose to fatty acid oxidation as the preferred metabolic substrate. Methods 30 critically ill patients (70% male, age 56.4 ± 19.7 years, APACHE II score 22.4 ± 6.6) were prospectively recruited Results There were reduction in both skeletal muscle mtDNA (p = 0.04) and PGC1-α (p = 0.02) from day 1 to day 7, with fold change in PGC1α correlated with the fold change in mtDNA (r 2 = 0.65, p 0.1). Protein levels of mitochondrial respiratory complexes I-V did not change significantly from day 1 to day 7 and mtDNA fold change did not correlate with fold change in complex I-V (p > 0.1). There was a weak positive correlation between feeding (protein delivered) and fold change in complex I (r 2 = 0.20, p = 0.014) and complex III protein level (r 2 = 0.20, p = 0.016). Overall levels of mitochondrial β-oxidation pathway proteins (CPT1, MCAD, ETF, DecR1) did not change whilst peroxisomal MEFII increased significantly (p Conclusion These data suggest decreased mitochondrial biogenesis over the first week of critical illness. The increase in β-oxidation proteins combined with the lack of change in respiratory chain complex concentrations during the first week suggests that the oxidative capacity of the remaining mitochondria was enhanced with increased capacity to metabolise fatty acids. These data support our hypothesis that the decreased cellular energy utilisation accompanying decreased mitochondrial biogenesis is offset by increased capacity to produce ATP through the β-oxidation of fats.

Apolipoprotein E4 Domain Interaction Mediates Detrimental Effects on Mitochondria and Is a Potential Therapeutic Target for Alzheimer Disease

Apolipoprotein (apo) E4 is the major genetic risk factor for late-onset Alzheimer disease (AD). ApoE4 assumes a pathological conformation through an intramolecular interaction mediated by Arg-61 in the amino-terminal domain and Glu-255 in the carboxyl-terminal domain, referred to as apoE4 domain interaction. Because AD is associated with mitochondrial dysfunction, we examined the effect of apoE4 domain interaction on mitochondrial respiratory function. Steady-state amounts of mitochondrial respiratory complexes were examined in neurons cultured from brain cortices of neuron-specific enolase promoter-driven apoE3 (NSE-apoE3) or apoE4 (NSE-apoE4) transgenic mice. All subunits of mitochondrial respiratory complexes assessed were significantly lower in NSE-apoE4 neurons compared with NSE-apoE3 neurons. However, no significant differences in levels of mitochondrial complexes were detected between astrocytes expressing different apoE isoforms driven by the glial fibrillary acidic protein promoter, leading to our conclusion that the effect of apoE4 is neuron specific. In neuroblastoma Neuro-2A (N2A) cells, apoE4 expression reduced the levels of mitochondrial respiratory complexes I, IV, and V. Complex IV enzymatic activity was also decreased, lowering mitochondrial respiratory capacity. Mutant apoE4 (apoE4-Thr-61) lacking domain interaction did not induce mitochondrial dysfunction in N2A cells, indicating that the effect is specific to apoE4-expressing cells and dependent on domain interaction. Consistent with this finding, treatment of apoE4-expressing N2A cells with a small molecule that disrupts apoE4 domain interaction restored mitochondrial respiratory complex IV levels. These results suggest that pharmacological intervention with small molecules that disrupt apoE4 domain interaction is a potential therapeutic approach for apoE4-carrying AD subjects.

Selected flavonoids potentiate the toxicity of cisplatin in human lung adenocarcinoma cells: A role for glutathione depletion

Adjuvant therapies that enhance the anti-tumor effects of cis-diammineplatinum(II) dichloride (cisplatin, CDDP) are actively being pursued. Growing evidence supports the involvement of mitochondrial dysfunction in the anti-cancer effect of cisplatin. We examined the potential of using selective flavonoids that are effective in depleting tumor cells of glutathione (GSH) to potentiate cisplatin-mediated cytotoxicity in human lung adenocarcinoma (A549) cells. We found that cisplatin (40 microM, 48-h treatment) disrupts the steady-state levels of mitochondrial respiratory complex I, which correlates with elevated mitochondrial reactive oxygen species (ROS) production and cytochrome c release. The flavonoids, 2',5'-dihydroxychalcone (2',5'-DHC, 20 microM) and chrysin (20 microM) potentiated the cytotoxicity of cisplatin (20 microM), which could be blocked by supplementation of the media with exogenous GSH (500 microM). Both 2',5'-DHC and chrysin were more effective than the specific inhibitor of GSH synthesis, L-buthionine sulfoximine (BSO, 20 microM), in inducing GSH depletion and potentiating the cytotoxic effect of cisplatin. These data suggest that the flavonoid-induced potentiation of cisplatin's toxicity is due, in part, to synergetic pro-oxidant effects of cisplatin by inducing mitochondrial dysfunction, and the flavonoids by depleting cellular GSH, an important antioxidant defense.

Akt signaling pathway in pacing-induced heart failure

Marked changes in energy substrate utilization occur during the progression of congestive heart failure (CHF) where fatty acid utilization, as the primary source of cardiac energy, is severely diminished, oxidative phosphorylation is down-regulated, and glucose uptake and utilization increase. Neither the signaling events or the molecular basis for the shift in substrate utilization have yet been elucidated. This study was designed to examine in the canine model of paced-induced CHF, the potential role of the Akt pathway in signaling the metabolic transitions central to progression to heart failure. Myocardial Akt levels were elevated in early heart failure (after 1-2 weeks of pacing) accompanied by increased levels of oxidative stress, cytokine tumor necrosis factor-alpha (TNF-alpha) and free fatty acid accumulation, reduced activity levels of mitochondrial respiratory complexes III and V and apoptosis initiation. At severe heart failure (3-4 weeks of pacing), there was significant further increase in myocardial apoptosis, with pronounced decline in myocardial Akt kinase activity. At this later stage, there were no further changes in free fatty acid accumulation, complex V activity or in oxidative stress levels indicating that these changes primarily occurred in the earlier stage of evolving heart failure. In contrast, during severe heart failure, both the reduction in complex III activity and increase in TNF-alpha level became more pronounced. Our data provide critical support for the hypothesis that the Akt signaling pathway is a contributory element in the early signaling events leading to the progression of pacing-induced heart failure, accompanying the shift in substrate utilization.