Modifications Of Mitochondrial Function Research Articles

Acid-base balance and long COVID: comments on metabolic-respiratory alterations

Long COVID is a multi-organ pathology with important sequelae that affect the health and welfare of survivors from the cellular bioenergetics, in this case, the importance of considering the acid-base balance within the processes of evaluation and treatment of long COVID by health and sports professionals was addressed, given that different investigations have found important modifications in mitochondrial function that result in ventilation failures, causing alterations in the compensation-decompensation of respiratory and renal pH control (metabolic-respiratory acidosis and alkalosis).

LMP1 mediates tumorigenesis through persistent epigenetic modifications and PGC1β upregulation.

Latent membrane protein 1 (LMP1), which is encoded by the Epstein‑Barr virus (EBV), has been considered as an oncogene, although the detailed mechanism behind its function remains unclear. It has been previously reported that LMP1 promotes tumorigenesis by upregulation of peroxisome proliferator‑activated receptor‑γ coactivator‑1β (PGC1β). The present study aimed to investigate the potential mechanism for transient EBV/LMP1 exposure‑mediated persistent PGC1β expression and subsequent tumorigenesis through modification of mitochondrial function. Luciferase reporter assay, chromatin immunoprecipitation and DNA mutation techniques were used to evaluate the PGC1β‑mediated expression of dynamin‑related protein 1 (DRP1). Tumorigenesis was evaluated by gene expression, oxidative stress, mitochondrial function and invitro cellular proliferation assays. The potential effects of EBV, LMP1 and PGC1β on tumor growth were evaluated in an invivo xenograft mouse model. The present invitro experiments showed that LMP1 knockdown did not affect PGC1β expression or subsequent cell proliferation in EBV‑positive tumor cells. PGC1β regulated DRP1 expression by coactivation of GA‑binding protein α and nuclear respiratory factor 1 located on the DRP1 promoter, subsequently modulating mitochondrial fission. Transient exposure of either EBV or LMP1 in human hematopoietic stem cells caused persistent epigenetic changes and PGC1β upregulation after long‑term cell culture even in the absence of EBV/LMP1, which decreased oxidative stress, and potentiated mitochondrial function and cell proliferation invitro. Enhanced tumor growth and shortened survival were subsequently observed invivo. It was concluded that PGC1β expression and subsequent cell proliferation were independent from LMP1 in EBV‑positive tumor cells. PGC1β modulated mitochondria fission by regulation of DRP1 expression. Transient EBV/LMP1 exposure caused persistent PGC1β expression, triggering tumor growth in the absence of LMP1. The present study proposes a novel mechanism for transient EBV/LMP1 exposure‑mediated tumorigenesis through persistent epigenetic changes and PGC1β upregulation, uncovering the reason why numerous forms of lymphoma exhibit upregulated PGC1β expression, but are devoid of EBV/LMP1.

Mitochondrial-targeting antioxidant MitoQ modulates angiogenesis and promotes functional recovery after spinal cord injury

BackgroundIn traumatic spinal cord injury (SCI), secondary injuries, including cellular death, mitochondrial dysfunction, and vascular injury, have been considered as important causes of impaired functional recovery after SCI. Postinjury angiogenesis has been considered to be a potential strategy for SCI treatment. New-born vessels may play a key role in nerve regeneration, which indicates the importance of angiogenesis in nerve regeneration. Recent studies have revealed the crosstalk between reactive oxygen species (ROS) and angiogenesis. As the main source of cellular ROS, mitochondria have been proven to be essential to the angiogenesis process. MethodsSCI was established in a T10 clip-compression animal model. Then, the animals received an intraperitoneal injection of MitoQ (5 mg/kg/d) on Days 0, 1, and 2 after surgery. The Basso Mouse Scale (BMS) score and footprint analysis (CatWalk analysis) were performed to evaluate functional recovery after SCI. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1/Neurofilament) were performed to evaluate angiogenesis, microglia activation and neural regeneration. RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) was performed to evaluate angiogenesis-related factor in injured spinal cord. ATP production assay and western-blotting assay (Mfn-1 and Drp-1) were performed to evaluate mitochondrial function in the injured spinal cord. BV2 cells were used as in vitro cell model. After receiving TBHP or TBHP-MitoQ treatment, ELISA and immunofluorescence assays were used to evaluate the level of VEGFA secretion from BV2 cells. A coculture system of HUVECs and BV2 cells was established. Tube formation assays and immunofluorescence assays (CD31) were performed on HUVECs in a coculture system to evaluate angiogenesis promotion. ATP production assays were performed to evaluate mitochondrial function in BV2 cells. MitoSOX Red and DCFH-DA staining were performed to evaluate mitochondrial and cellular ROS. ResultsIn vitro MitoQ promoted the secretion of VEGFA from BV2 cells, which was verified through ELISA and immunofluorescence assays. The angiogenic promotion of MitoQ-treated BV2 cells was evaluated by tube formation and immunofluorescence assays (CD31) in a coculture system of BV2 cells and HUVECs. MitoQ inhibited cellular and mitochondrial-derived ROS in TBHP-treated BV2 cells. ATP production was increased in MitoQ-treated BV2 cells. To verify MitoQ’s effect in vivo, a T10 clip-compression animal model was established successfully. MitoQ significantly promoted functional recovery, as shown by the BMS assay and gait analysis. The promotion of neural regeneration was identified through immunofluorescence assay of neurofilament. Immunofluorescence and fluorescence assays (LEL-FITC/CD31/Iba-1) and RT-qPCR (VEGFR-1, VEGFR-2 and VEGFA) indicated that MitoQ could promote angiogenesis and inhibit macrophage/microglia activation in lesion-site after SCI. Enhanced ATP production and increased Mfn-1 with decreased Drp-1 protein expression showed MitoQ could promote mitochondrial function in SCI. ConclusionThe mitochondrial-specific antioxidant MitoQ promotes functional recovery and tissue preservation through the enhancement of angiogenesis with the modification of mitochondrial function after SCI.

Atorvastatin impairs liver mitochondrial function in obese G\xf6ttingen Minipigs but heart and skeletal muscle are not affected

Statins lower the risk of cardiovascular events but have been associated with mitochondrial functional changes in a tissue-dependent manner. We investigated tissue-specific modifications of mitochondrial function in liver, heart and skeletal muscle mediated by chronic statin therapy in a Göttingen Minipig model. We hypothesized that statins enhance the mitochondrial function in heart but impair skeletal muscle and liver mitochondria. Mitochondrial respiratory capacities, citrate synthase activity, coenzyme Q10 concentrations and protein carbonyl content (PCC) were analyzed in samples of liver, heart and skeletal muscle from three groups of Göttingen Minipigs: a lean control group (CON, n = 6), an obese group (HFD, n = 7) and an obese group treated with atorvastatin for 28 weeks (HFD + ATO, n = 7). Atorvastatin concentrations were analyzed in each of the three tissues and in plasma from the Göttingen Minipigs. In treated minipigs, atorvastatin was detected in the liver and in plasma. A significant reduction in complex I + II-supported mitochondrial respiratory capacity was seen in liver of HFD + ATO compared to HFD (P = 0.022). Opposite directed but insignificant modifications of mitochondrial respiratory capacity were seen in heart versus skeletal muscle in HFD + ATO compared to the HFD group. In heart muscle, the HFD + ATO had significantly higher PCC compared to the HFD group (P = 0.0323). In the HFD group relative to CON, liver mitochondrial respiration decreased whereas in skeletal muscle, respiration increased but these changes were insignificant when normalizing for mitochondrial content. Oral atorvastatin treatment in Göttingen Minipigs is associated with a reduced mitochondrial respiratory capacity in the liver that may be linked to increased content of atorvastatin in this organ.

Phosphorylation of mitochondrial transcription factor B2 controls mitochondrial DNA binding and transcription

Mammalian cells contain genetic information in two compartments, the nucleus and the mitochondria. Mitochondrial gene expression must be coordinated with nuclear gene expression to respond to cellular energetic needs. To gain insight into the coordination between the nucleus and mitochondria, there is a need to understand the regulation of transcription of mitochondrial DNA (mtDNA). Reversible protein post-translational modifications of the mtDNA transcriptional machinery may be one way to control mtDNA transcription. Here we focus on a member of the mtDNA transcription initiation complex, mitochondrial transcription factor B2 (TFB2M). TFB2M melts mtDNA at the promoter to allow the RNA polymerase (POLRMT) to access the DNA template and initiate transcription. Three phosphorylation sites have been previously identified on TFB2M by mass spectrometry: threonine 184, serine 197, and threonine 313. Phosphomimetics were established at these positions. Proteins were purified and analyzed for their ability to bind mtDNA and initiate transcription in vitro. Our results indicate phosphorylation at threonine 184 and threonine 313 impairs promoter binding and prevents transcription. These findings provide a potential regulatory mechanism of mtDNA transcription and help clarify the importance of protein post-translational modifications in mitochondrial function.

Altered mitochondrial oxidative phosphorylation capacity in horses suffering from polysaccharide storage myopathy.

Polysaccharide storage myopathy (PSSM) is a widely described cause of exertional rhabdomyolysis in horses. Mitochondria play a central role in cellular energetics and are involved in human glycogen storage diseases but their role has been overlooked in equine PSSM. We hypothesized that the mitochondrial function is impaired in the myofibers of PSSM-affected horses. Nine horses with a history of recurrent exercise-associated rhabdomyolysis were tested for the glycogen synthase 1 gene (GYS1) mutation: 5 were tested positive (PSSM group) and 4 were tested negative (horses suffering from rhabdomyolysis of unknown origin, RUO group). Microbiopsies were collected from the gluteus medius (gm) and triceps brachii (tb) muscles of PSSM, RUO and healthy controls (HC) horses and used for histological analysis and for assessment of oxidative phosphorylation (OXPHOS) using high-resolution respirometry. The modification of mitochondrial respiration between HC, PSSM and RUO horses varied according to the muscle and to substrates feeding OXPHOS. In particular, compared to HC horses, the gm muscle of PSSM horses showed decreased OXPHOS- and electron transfer (ET)-capacities in presence of glutamate&malate&succinate. RUO horses showed a higher OXPHOS-capacity (with glutamate&malate) and ET-capacity (with glutamate&malate&succinate) in both muscles in comparison to the PSSM group. When expressed as ratios, our results highlighted a higher contribution of the NADH pathway (feeding electrons into Complex I) to maximal OXPHOS or ET-capacity in both rhabdomyolysis groups compared to the HC. Specific modifications in mitochondrial function might contribute to the pathogenesis of PSSM and of other types of exertional rhabdomyolyses.

Modification of mitochondrial function, cytoplasmic lipid content and cryosensitivity of bovine embryos by resveratrol.

Resveratrol is a potent activator of NAD-dependent deacetyltransferase sirtuin-1 (SIRT1) and affects lipid metabolism and ATP generation in somatic cells. In the present study, the effects of supplementing culture medium with resveratrol on lipid metabolism, ATP generation, and cryosensitivity of bovine in vitro produced embryos were investigated. Bovine early cleaved-stage embryos were cultured in medium containing 0 or 0.5 µM resveratrol for 1 or 5 days. Resveratrol treatment for both 1 day and 5 days increased the expression levels of SIRT1 and phosphorylated AMP-activated protein kinase (pAMPK) in the embryos. Furthermore, resveratrol treatment was effective to increase ATP generation and reduce lipid content of the embryos. The effects of resveratrol treatment were diminished by the SIRT1 inhibitor “EX527”, and the reduced lipid content was reversed by treatment with etomoxir (a potent inhibitor of beta-oxidation). Blastocysts developed after resveratrol treatment showed low levels reactive oxygen species and increased cryotolerance. These results demonstrate that resveratrol improves in vitro development of bovine embryos, while reducing cytoplasmic lipid content through activation of beta-oxidation, thereby effective for production of bovine blastocysts with enhanced cryotolerance.

Postconditioning inhibits mPTP opening independent of oxidative phosphorylation and membrane potential

Mitochondrial permeability transition pore (mPTP) inhibition plays a relevant role in postconditioning (PostC). Ischemia damages the electron transport chain, and the potential contribution of additional modifications in mitochondrial function caused by PostC remains unknown. We sought to determine which mitochondrial functions are involved in the inhibition of mPTP opening during the first minutes of reperfusion. Anesthetized New Zealand White rabbits underwent 30-min ischemia followed by 10-min reperfusion. At reperfusion, they received either no intervention (Control, C), PostC with 4 cycles of 1-min ischemia followed by 1-min reperfusion, or an IV injection of 5 mg/kg cyclosporine A (CsA: a powerful inhibitor of mPTP opening). Sham rabbits underwent no ischemia throughout the 40-min experiment. At the end of the 10-min reperfusion, mitochondria were isolated from the area at risk by differential centrifugations. Calcium retention capacity (CRC) and mitochondrial membrane potential (Δ Ψ m) were assessed by fluorimetry in subsarcolemmal (SSM) and interfibrillar (IFM) mitochondria. Oxidative phosphorylation was assessed using a Clark-type electrode, and oxidative stress via protein carbonylation by Western blotting. PostC and CsA treatments improved CRC when compared to the C group. Control, PostC and CsA mitochondria exhibited a comparable significant dissipation of Δ Ψ m, together with a comparable significant decrease in state 3 and an increase in state 4 respiration, in both SSM and IFM. However, PostC but not CsA treatment reduced total heart oxidative stress. These data suggest that during the early minutes of reperfusion, PostC reduces oxidative stress and inhibits mPTP opening, independent of alteration of oxidative phosphorylation or of Δ Ψ m.

Spatially resolved fluorescence lifetime mapping of enzyme kinetics in living cells

Traditional cuvette-based enzyme studies lack spatial information and do not allow real-time monitoring of the effects of modulating enzyme functions in vivo. In order to probe the realistic timescales of steric modifications in enzyme-substrate complexes and functional binding-unbinding kinetics in living cells without losing spatial information, it is imperative to develop sensitive imaging strategies that can report enzyme kinetics in real time over a wide dynamic range of timescales. Here we present a multi-photon excitation-based, ultra-fast photon detection using a streak camera and Laguerre expansion-based fast deconvolution approach for achieving high spatio-temporal resolution in monitoring real-time enzyme kinetics in single cells. In particular, we report spatially resolved, nanosecond-scale fluorescence dynamics associated with binding-unbinding kinetics of endogenous metabolic co-factor nicotinamide adenine dinucleotide with enzymes in intact living cells. By monitoring real-time kinetics of NAD(P)H-enzyme kinetics in primary hepatocytes isolated from young and aged mouse models, we observed that the mechanism of inhibition of mitochondrial respiration at complex I site is mediated by redistribution of free and protein-bound nicotinamide adenine dinucleotide pools and that this equilibrium redistribution is affected by age-related modifications in mitochondrial function. We describe unique advantages of Laguerre deconvolution algorithm in comparison with conventional lifetime analysis approaches. Non-invasive monitoring of metabolic dysfunctions in intact animal models is an attractive strategy for gaining insight into the dynamics of tissue metabolism in health and in various metabolic syndromes such as cancer, diabetes and aging-induced metabolic dysfunctions. Besides the example demonstrated above, we envisage that the proposed method can find applications in a variety of other situations where intensity-based approaches fall short owing to spectroscopic artefacts.

Mitochondrial Matrix Phosphoproteome: Effect of Extra Mitochondrial Calcium

Post-translational modification of mitochondrial proteins by phosphorylation or dephosphorylation plays an essential role in numerous cell signaling pathways involved in regulating energy metabolism and in mitochondrion-induced apoptosis. Here we present a phosphoproteomic screen of the mitochondrial matrix proteins and begin to establish the protein phosphorylations acutely associated with calcium ions (Ca(2+)) signaling in porcine heart mitochondria. Forty-five phosphorylated proteins were detected by gel electrophoresis-mass spectrometry of Pro-Q Diamond staining, while many more Pro-Q Diamond-stained proteins evaded mass spectrometry detection. Time-dependent (32)P incorporation in intact mitochondria confirmed the extensive matrix protein phosphoryation and revealed the dynamic nature of this process. Classes of proteins that were detected included all of the mitochondrial respiratory chain complexes, as well as enzymes involved in intermediary metabolism, such as pyruvate dehydrogenase (PDH), citrate synthase, and acyl-CoA dehydrogenases. These data demonstrate that the phosphoproteome of the mitochondrial matrix is extensive and dynamic. Ca(2+) has previously been shown to activate various dehydrogenases, promote the generation of reactive oxygen species (ROS), and initiate apoptosis via cytochrome c release. To evaluate the Ca(2+) signaling network, the effects of a Ca(2+) challenge sufficient to release cytochrome c were evaluated on the mitochondrial phosphoproteome. Novel Ca(2+)-induced dephosphorylation was observed in manganese superoxide dismutase (MnSOD) as well as the previously characterized PDH. A Ca(2+) dose-dependent dephosphorylation of MnSOD was associated with an approximately 2-fold maximum increase in activity; neither the dephosphorylation nor activity changes were induced by ROS production in the absence of Ca(2+). These data demonstrate the use of a phosphoproteome screen in determining mitochondrial signaling pathways and reveal new pathways for Ca(2+) modification of mitochondrial function at the level of MnSOD.

A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center

Mitochondria in the oxidizing environment of the maize (Zea mays) root quiescent center (QC) are altered in function, but otherwise structurally normal. Compared to mitochondria in the adjacent, rapidly dividing cells of the proximal root tissues, mitochondria in the QC show marked reductions in the activities of tricarboxylic acid cycle enzymes. Pyruvate dehydrogenase activity was not detected in the QC. Use of several mitochondrial membrane potential (DeltaPsi(m)) sensing probes indicated a depolarization of the mitochondrial membrane in the QC, which suggests a reduction in the capacity of QC mitochondria to generate ATP and NADH. We postulate that modifications of mitochondrial function are central to the establishment and maintenance of the QC.

Effect of vitamin B6 on oxygen radicals, mitochondrial membrane potential, and lipid peroxidation in H2O2-treated U937 monocytes

Vitamin B6 (Vit.B6) supplementation has been shown to be beneficial in reducing diabetic complications, cognitive aging, and in the prevention of coronary heart disease. It was hypothesized that Vit.B6 compounds may function as antioxidants and thus offer protection against oxidative stress under various pathophysiological and or experimental conditions. To test this hypothesis, U937 monocytes were cultured with pyridoxine (P), pyridoxal phosphate (PP) and pyridoxamine (PM) and H2O2, either alone or together for 2 h. Oxidative stress was determined by measuring superoxide radical production, lipid peroxidation, and mitochondrial transmembrane potential. Results demonstrate that Vit.B6 compounds can prevent the oxygen radical generation and lipid peroxidation caused by hydrogen peroxide in U937 monocytes, and that some of the protective effect of Vit.B6 may occur via modification of mitochondrial function.

In situ mitochondrial function in volume overload- and pressure overload-induced cardiac hypertrophy in rats.

Little comparative information is available on mitochondrial function changes during experimentally-induced hypertrophy. Respiratory control mechanisms are not exactly the same in situ and in isolated mitochondria. This study assessed in situ mitochondrial function in two myocardial hypertrophy models. Cytochrome aa3 (Cytaa3) and myoglobin (Mb) absorption changes were monitored in isolated rat hearts using dual wavelength spectrophotometry (Cytaa3: 605-630 nm, Mb: 581-592 nm). Hypertrophy was induced by creation of an aortic stenosis or of an aorto-caval fistula. Optical monitoring was performed on diastole-arrested perfused hearts using the sequence O2 perfusion, N2 perfusion during 4 min, and reoxygenation. The plateaus of the Cytaa3 and Mb curves were used to quantify oxidation-reduction and oxygenation levels. Respiratory kinetics were characterized by the slopes of transition phase curves. Myoglobin oxygenation was comparable in the hypertrophied and control hearts. However, Cytaa3 oxidation-reduction levels in the hypertrophied hearts showed a shift towards greater reduction in comparison with the controls (controls: 0.580 +/- 0.008 DO605/DO630 nm, n = 34; fistula: 0.530 +/- 0.023, n = 23; stenosis: 0.522 +/- 0.016, n = 20, p < 0.001). The rate of Cytaa3 reduction and the rate of myoglobin deoxygenation were significantly accelerated (p < 0.005) in the volume overload group (0.507 +/- 0.043, n = 23), whereas the respiratory rate in the pressure overload group (0.389 +/- 0.034, n = 20) was comparable to that in the control hearts (0.358 +/- 0.026 delta DO 605 nm/DO630 nm.min-1, n = 34). We found mitochondrial function alterations in both volume overload- and pressure overload-induced cardiac hypertrophy, despite adequate cytosol oxygenation. The patterns of these alterations differed: the redox state showed a shift of similar magnitude toward greater reduction in both models, but the respiratory rate was increased in the volume-overloaded hearts and unchanged in the pressure-overloaded hearts. The modification in the oxidation-reduction state suggested that overload hypertrophy may induce changes in the metabolism of the myocardium, which may, in turn, load to persistent modifications in mitochondrial function. The differences between the two models suggest that adaptation to hypertrophy-inducing events exists at the level of the mitochondrion.