High resolution respirometry analysis of polyethylenimine-mediated mitochondrial energy crisis and cellular stress: Mitochondrial proton leak and inhibition of the electron transport system

Polyethylenimines (PEIs) are highly efficient non-viral transfectants, but can induce cell death through poorly understood necrotic and apoptotic processes as well as autophagy. Through high resolution respirometry studies in H1299 cells we demonstrate that the 25 kDa branched polyethylenimine (25k-PEI-B), in a concentration and time-dependent manner, facilitates mitochondrial proton leak and inhibits the electron transport system. These events were associated with gradual reduction of the mitochondrial membrane potential and mitochondrial ATP synthesis. The intracellular ATP levels further declined as a consequence of PEI-mediated plasma membrane damage and subsequent ATP leakage to the extracellular medium. Studies with freshly isolated mouse liver mitochondria corroborated with bioenergetic findings and demonstrated parallel polycation concentration- and time-dependent changes in state 2 and state 4o oxygen flux as well as lowered ADP phosphorylation (state 3) and mitochondrial ATP synthesis. Polycation-mediated reduction of electron transport system activity was further demonstrated in ‘broken mitochondria’ (freeze-thawed mitochondrial preparations). Moreover, by using both high-resolution respirometry and spectrophotometry analysis of cytochrome c oxidase activity we were able to identify complex IV (cytochrome c oxidase) as a likely specific site of PEI mediated inhibition within the electron transport system. Unraveling the mechanisms of PEI-mediated mitochondrial energy crisis is central for combinatorial design of safer polymeric non-viral gene delivery systems.     

Using exomarkers to assess mitochondrial reactive species in vivo

Background

The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.

Scope of review

One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.

Major conclusions and general significance

Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.     

Cationic antioxidants as a powerful tool against mitochondrial oxidative stress

This review describes evidence that mitochondrial reactive oxygen species (mROS) are of great importance under many physiological and pathological conditions. The most demonstrative indications favoring this conclusion originate from recent discoveries of the in vivo effects of mitochondria-targeted antioxidants (MitoQ and SkQs). The latter compounds look promising in treating several incurable pathologies as well as aging.     

Comparison of static and dynamic respirometry for the determination of stoichiometric and kinetic parameters of a nitrifying process

Respirometry consists in the measurement of the biological oxygen consumption rate under well-defined conditions and has been used for the characterization of countless biological processes. In the field of biotechnology and applied microbiology, several respirometry methods are commonly used for the determination of process parameters. Dynamic and static respirometry, which are based on oxygen measurements with or without continuous aeration, respectively, are the methods most commonly used. Additionally to several respirometry methods, different methods have also been developed to retrieve process parameters from respirometric data. Among them, methods based on model fitting and methods based on the injection of substrate pulse at increasing concentration are commonly used. An important question is then; what respirometry and data interpretation methods should be preferably used? So far, and despite a growing interest for respirometry, relatively little attention has been paid on the comparison between the different methods available. In this work, both static and dynamic respirometry methods and both interpretation methods; model fitting and pulses of increasing concentration, were compared to characterize an autotrophic nitrification process. A total of 60 respirometry experiments were done and exhaustively analysed, including sensitivity and error analyses. According to the results obtained, the substrate affinity constant (K _S ) was better determined by static respirometry with pulses of increasing concentration and the maximum oxygen uptake rate (OUR _ex.max ) was better determined by dynamic respirometry coupled to fitting procedure. The best method for combined K _S and OUR _ex.max determination was static respirometry with pulses of increasing concentration.     

Trace amounts of 8-oxo-dGTP in mitochondrial dNTP pools reduce DNA polymerase gamma replication fidelity

Replication of the mitochondrial genome by DNA polymerase γ requires dNTP precursors that are subject to oxidation by reactive oxygen species generated by the mitochondrial respiratory chain. One such oxidation product is 8-oxo-dGTP, which can compete with dTTP for incorporation opposite template adenine to yield A-T to C-G transversions. Recent reports indicate that the ratio of undamaged dGTP to dTTP in mitochondrial dNTP pools from rodent tissues varies from ~1:1 to >100:1. Within this wide range, we report here the proportion of 8-oxo-dGTP in the dNTP pool that would be needed to reduce the replication fidelity of human DNA polymerase γ. When various in vivo mitochondrial dNTP pools reported previously were used here in reactions performed in vitro, 8-oxo-dGTP was readily incorporated opposite template A and the resulting 8-oxo-G-A mismatch was not proofread efficiently by the intrinsic 3′ exonuclease activity of pol γ. At the dNTP ratios reported in rodent tissues, whether highly imbalanced or relatively balanced, the amount of 8-oxo-dGTP needed to reduce fidelity was <1% of dGTP. Moreover, direct measurements reveal that 8-oxo-dGTP is present at such concentrations in the mitochondrial dNTP pools of several rat tissues. The results suggest that oxidized dNTP precursors may contribute to mitochondrial mutagenesis in vivo, which could contribute to mitochondrial dysfunction and disease.     

A high-throughput respirometric assay for mitochondrial biogenesis and toxicity

Mitochondria are a common target of toxicity for drugs and other chemicals and result in decreased aerobic metabolism and cell death. In contrast, mitochondrial biogenesis restores cell vitality, and there is a need for new agents to induce biogenesis. Current cell-based models of mitochondrial biogenesis or toxicity are inadequate because cultured cell lines are highly glycolytic with minimal aerobic metabolism and altered mitochondrial physiology. In addition, there are no high-throughput real-time assays that assess mitochondrial function. We adapted primary cultures of renal proximal tubular cells (RPTCs) that exhibit in vivo levels of aerobic metabolism, are not glycolytic, and retain higher levels of differentiated functions and used the Seahorse Bioscience analyzer to measure mitochondrial function in real time in multiwell plates. Using uncoupled respiration as a marker of electron transport chain (ETC) integrity, the nephrotoxicants cisplatin, HgCl₂, and gentamicin exhibited mitochondrial toxicity prior to decreases in basal respiration and cell death. Conversely, using FCCP (carbonylcyanide p-trifluoromethoxyphenylhydrazone)-uncoupled respiration as a marker of maximal ETC activity, 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI), SRT1720, resveratrol, daidzein, and metformin produced mitochondrial biogenesis in RPTCs. The merger of the RPTC model and multiwell respirometry results in a single high-throughput assay to measure mitochondrial biogenesis and toxicity and nephrotoxic potential.     

The yeast mitochondrial succinylome: Implications for regulation of mitochondrial nucleoids

Acylation modifications, such as the succinylation of lysine, are post-translational modifications and a powerful means of regulating protein activity. Some acylations occur nonenzymatically, driven by an increase in the concentration of acyl group donors. Lysine succinylation has a profound effect on the corresponding site within the protein, as it dramatically changes the charge of the residue. In eukaryotes, it predominantly affects mitochondrial proteins because the donor of succinate, succinyl-CoA, is primarily generated in the tricarboxylic acid cycle. Although numerous succinylated mitochondrial proteins have been identified in Saccharomyces cerevisiae, a more detailed characterization of the yeast mitochondrial succinylome is still lacking. Here, we performed a proteomic MS analysis of purified yeast mitochondria and detected 314 succinylated mitochondrial proteins with 1763 novel succinylation sites. The mitochondrial nucleoid, a complex of mitochondrial DNA and mitochondrial proteins, is one of the structures whose protein components are affected by succinylation. We found that Abf2p, the principal component of mitochondrial nucleoids responsible for compacting mitochondrial DNA in S. cerevisiae, can be succinylated in vivo on at least thirteen lysine residues. Abf2p succinylation in vitro inhibits its DNA-binding activity and reduces its sensitivity to digestion by the ATP-dependent ScLon protease. We conclude that changes in the metabolic state of a cell resulting in an increase in the concentration of tricarboxylic acid intermediates may affect mitochondrial functions.     

Control of mitochondrial and cellular respiration by oxygen

Control and regulation of mitochondrial and cellular respiration by oxygen is discussed with three aims: (1) A review of intracellular oxygen levels and gradients, particularly in heart, emphasizes the dominance of extracellular oxygen gradients. Intracellular oxygen pressure, $p_{O_2 } $ , is low, typically one to two orders of magnitude below incubation conditions used routinely for the study of respiratory control in isolated mitochondria. The $p_{O_2 } $ range of respiratory control by oxygen overlaps with cellular oxygen profiles, indicating the significance of $p_{O_2 } $ in actual metabolic regulation. (2) A methodologically detailed discussion of high-resolution respirometry is necessary for the controversial topic of respiratory control by oxygen, since the risk of methodological artefact is closely connected with far-reaching theoretical implications. Instrumental and analytical errors may mask effects of energetic state and partially explain the divergent views on the regulatory role of intracellular $p_{O_2 } $ . Oxygen pressure for half-maximum respiration,p ₅₀, in isolated mitochondria at state 4 was 0.025 kPa (0.2 Torr; 0.3 ΜM O₂), whereasp ₅₀ in endothelial cells was 0.06–0.08 kPa (0.5 Torr). (3) A model derived from the thermodynamics of irreversible processes was developed which quantitatively accounts for near-hyperbolic flux/ $p_{O_2 } $ relations in isolated mitochondria. The apparentp ₅₀ is a function of redox potential and protonmotive force. The protonmotive force collapses after uncoupling and consequently causes a decrease inp ₅₀. Whereas it is becoming accepted that flux control is shared by several enzymes, insufficient attention is paid to the notion of complementary kinetic and thermodynamic flux control mechanisms.     

Mitofusin 2 ameliorates hypoxia-induced apoptosis via mitochondrial function and signaling pathways

Mitochondrial dynamics play a critical role in mitochondrial function and signaling. Although mitochondria play a critical role in hypoxia/ischemia, the further mechanisms between mitochondrial dynamics and ischemia are still unclear. The current study aimed to determine the role of mitofusin 2, a key regulator of mitochondrial fusion, in a hypoxic model and to explore a novel strategy for cerebral ischemia via modulation of mitochondrial dynamics. To the best of our knowledge, this is the first study to investigate both mitochondrial function and molecular pathways to determine the role of mitofusin 2 in hypoxia-induced neuronal apoptosis. In vivo, C57BL/6 mice (male, 19–25 g) underwent a permanent middle cerebral artery occlusion for 12 or 24 h (n = 6 per group). In vitro, cobalt chloride was used to mimic hypoxia in immortalized hippocampal neurons. Down- or up-regulation of Mfn2 was induced to investigate the role of Mfn2 in hypoxia, especially in mitochondrial function and signaling pathways. The findings demonstrated that decreased mitofusin 2 occurred both in vivo and in vitro hypoxic models; second, the anti-apoptotic effect of Mfn2 may work via restoration of mitochondrial function; third, the modulation of the B Cell Leukemia 2/Bcl-2 Associated X protein and extracellular signal-regulated kinase 1/2 signaling pathways highlight the role of Mfn2 in signaling pathways beyond fusion. In summary, depletion of mitofusin 2 would lead to apoptosis both in normal or hypoxic conditions; however, mitofusin 2 overexpression could attenuate hypoxia-induced apoptosis, which represents a potential novel strategy for neuroprotection against ischemic brain damage.     

Interaction of NDPK-D with cardiolipin-containing membranes: Structural basis and implications for mitochondrial physiology

Nucleoside diphosphate kinases (NDPKs/Nm23), responsible for intracellular di- and tri-phosphonucleoside homeostasis, play multi-faceted roles in cellular energetic, signaling, proliferation, differentiation and tumor invasion. The mitochondrial NDPK-D, the NME4 gene product, is a peripheral protein of the inner membrane. Several new aspects of the interaction of NDPK-D with the inner mitochondrial membrane have been recently characterized. Surface plasmon resonance analysis using recombinant NDPK-D and different phospholipid liposomes showed that NDPK-D interacts electrostatically with anionic phospholipids, with highest affinity observed for cardiolipin, a phospholipid located mostly in the mitochondrial inner membrane. Mutation of the central arginine (R90) in a surface exposed cationic RRK motif unique to NDPK-D strongly reduced phospholipid interaction in vitro and in vivo. Stable expression of NDPK-D proteins in HeLa cells naturally almost devoid of this isoform revealed a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the enzyme. Owing to its symmetrical hexameric structure exposing membrane binding motifs on two opposite sides, NDPK-D could bridge liposomes containing anionic phospholipids and promote lipid transfer between them. In vivo, NDPK-D could induce intermembrane contacts and facilitate lipid movements between mitochondrial membranes. Most of these properties are reminiscent to those of the mitochondrial creatine kinase. We review here the common properties of both kinases and we discuss their potential roles in mitochondrial functions such as energy production, apoptosis and mitochondrial dynamics.     

A novel oxyconforming response in the freshwater fish Galaxias maculatus

How fish oxygen consumption is modulated by external PO₂ has long been a matter of interest, yet is an experimentally complicated question to answer. In this study closed and semi-closed respirometry were used to evaluate the oxygen consumption rate of the scaleless galaxiid fish, inanga (Galaxias maculatus) as a function of decreasing external PO₂. Both respirometry techniques showed that as environmental oxygen levels declined, oxygen consumption rates also decreased. At no point did inanga regulate oxygen consumption. This is strong evidence that inanga is an oxyconformer. Partitioned respirometry experiments showed that skin plays an important role in oxygen uptake in this fish species, and cutaneous oxygen uptake may have an important role in shaping the oxygen consumption response to hypoxia.     

Utilizing mitochondrial events as biomarkers for imaging apoptosis

Cells undergoing apoptosis show a plethora of time-dependent changes. The available tools for imaging apoptosis in live cells rely either on the detection of the activity of caspases, or on the visualization of exposure of phosphatidyl serine in the outer leaflet of the cell membrane. We report here a novel method for the detection of mitochondrial events during apoptosis, namely translocation of Bax to mitochondria and release of cytochrome c (Cyt c) using bimolecular fluorescence complementation. Expression of split yellow fluorescent protein (YFP) fragments fused to Bax and Cyt c, resulted in robust induction of YFP fluorescence at the mitochondria of apoptotic cells with very low background. In vivo expression of split YFP protein fragments in liver hepatocytes and intra-vital imaging of subcutaneous tumor showed elevated YFP fluorescence upon apoptosis induction. Thus, YFP complementation could be applied for high-throughput screening and in vivo molecular imaging of mitochondrial events during apoptosis.     

Drosophila RNase Z processes mitochondrial and nuclear pre-tRNA 3' ends in vivo

Although correct tRNA 3′ ends are crucial for protein biosynthesis, generation of mature tRNA 3′ ends in eukaryotes is poorly understood and has so far only been investigated in vitro. We report here for the first time that eukaryotic tRNA 3′ end maturation is catalysed by the endonuclease RNase Z in vivo. Silencing of the JhI-1 gene (RNase Z homolog) in vivo with RNAi in Drosophila S2 cultured cells causes accumulation of nuclear and mitochondrial pre-tRNAs, suggesting that JhI-1 encodes both forms of the tRNA 3′ endonuclease RNase Z, and establishing its biological role in endonucleolytic tRNA 3′ end processing. In addition our data show that in vivo 5′ processing of nuclear and mitochondrial pre-tRNAs occurs before 3′ processing.     

A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts

A potent analog (HNG) of the endogenous peptide humanin protects against myocardial ischemia–reperfusion (MI–R) injury in vivo, decreasing infarct size and improving cardiac function. Since oxidative stress contributes to the damage from MI–R we tested the hypotheses that: (1) HNG offers cardioprotection through activation of antioxidant defense mechanisms leading to preservation of mitochondrial structure and that, (2) the activity of either of a pair of non-receptor tyrosine kinases, c-Abl and Arg is required for this protection. Rat cardiac myoblasts (H₉C₂ cells) were exposed to nanomolar concentrations of HNG and to hydrogen peroxide (H₂O₂). Cells treated with HNG in the presence of H₂O₂ demonstrated reduced intracellular reactive oxygen species (ROS), preserved mitochondrial membrane potential, ATP levels and mitochondrial structure. HNG induced activation of catalase and glutathione peroxidase (GPx) within 5 min and decreased the ratio of oxidized to reduced glutathione within 30 min. siRNA knockdown of both Abl and Arg, but neither alone, abolished the HNG-mediated reduction of ROS in myoblasts exposed to H₂O₂. These findings demonstrate an HNG-mediated, Abl- and Arg-dependent, rapid and sustained activation of critical cellular defense systems and attenuation of oxidative stress, providing mechanistic insights into the observed HNG-mediated cardioprotection in vivo.     

Xanthohumol induces generation of reactive oxygen species and triggers apoptosis through inhibition of mitochondrial electron transfer chain complex I

Xanthohumol is a prenylflavonoid extracted from hops (Humulus lupulus). It possesses anti-cancer and anti-inflammatory activities in vitro and in vivo, and offers therapeutic benefits for treatment of metabolic syndromes. However, the precise mechanisms underlying its pharmacological effects remain to be elucidated, together with its cellular target. Here, we provide evidence that xanthohumol directly interacts with the mitochondrial electron transfer chain complex I (NADH dehydrogenase), inhibits the oxidative phosphorylation, triggers the production of reactive oxygen species, and induces apoptosis. In addition, we show that as a result of the inhibition of the mitochondrial oxidative phosphorylation, xanthohumol exposure causes a rapid decrease of mitochondrial transmembrane potential. Furthermore, we showed that xanthohumol up-regulates the glycolytic capacity in cells, and thus compensates cellular ATP generation. Dissection of the multiple steps of aerobic respiration by extracellular flux assays revealed that xanthohumol specifically inhibits the activity of mitochondrial complex I, but had little effect on that of complex II, III and IV. Inhibition of complex I by xanthohumol caused the overproduction of reactive oxygen species, which are responsible for the induction of apoptosis in cancer cells. We also found that isoxanthohumol, the structural isomer of xanthohumol, is inactive to cells, suggesting that the reactive 2-hydroxyl group of xanthohumol is crucial for its targeting to the mitochondrial complex I. Together, the remodeling of cell metabolism revealed here has therapeutic potential for the use of xanthohumol.     

POS5 gene of Saccharomyces cerevisiae encodes a mitochondrial NADH kinase required for stability of mitochondrial DNA

In a search for nuclear genes that affect mutagenesis of mitochondrial DNA in Saccharomyces cerevisiae, an ATP-NAD (NADH) kinase, encoded by POS5, that functions exclusively in mitochondria was identified. The POS5 gene product was overproduced in Escherichia coli and purified without a mitochondrial targeting sequence. A direct biochemical assay demonstrated that the POS5 gene product utilizes ATP to phosphorylate both NADH and NAD⁺, with a twofold preference for NADH. Disruption of POS5 increased minus-one frameshift mutations in mitochondrial DNA 50-fold, as measured by the arg8^m reversion assay, with no increase in nuclear mutations. Also, a dramatic increase in petite colony formation and slow growth on glycerol or limited glucose were observed. POS5 was previously described as a gene required for resistance to hydrogen peroxide. Consistent with a role in the mitochondrial response to oxidative stress, a pos5 deletion exhibited a 28-fold increase in oxidative damage to mitochondrial proteins and hypersensitivity to exogenous copper. Furthermore, disruption of POS5 induced mitochondrial biogenesis as a response to mitochondrial dysfunction. Thus, the POS5 NADH kinase is required for mitochondrial DNA stability with a critical role in detoxification of reactive oxygen species. These results predict a role for NADH kinase in human mitochondrial diseases.     

Cutaneous Respirometry as Novel Technique to Monitor Mitochondrial Function: A Feasibility Study in Healthy Volunteers

BackgroundThe protoporphyrin IX-triplet state lifetime technique (PpIX-TSLT) is proposed as a potential clinical non-invasive tool to monitor mitochondrial function. This technique has been evaluated in several animal studies. Mitochondrial respirometry allows measurement in vivo of mitochondrial oxygen tension (mitoPO₂) and mitochondrial oxygen consumption (mitoVO₂) in skin. This study describes the first use of a clinical prototype in skin of humans.MethodsThe clinical prototype was tested in 30 healthy volunteers. A self-adhesive patch containing 2 mg 5-aminolevulinic acid (ALA) was applied on the skin of the anterior chest wall (sternal) for induction of mitochondrial protoporphyrin IX and was protected from light for 5 h. MitoPO₂ was measured by means of oxygen-dependent delayed fluorescence of protoporphyrin IX. MitoVO₂ was determined by dynamic mitoPO₂ measurements on the primed skin, while locally blocking oxygen supply by applying local pressure with the measurement probe. MitoPO₂ was recorded before and during a 60-s period of compression of the microcirculation, at an interval of 1 Hz. Oxygen consumption (i.e. the local oxygen disappearance rate) was calculated from the decay of the mitoPO₂ slope.ResultsOxygen-dependent delayed fluorescence measurements were successfully performed in the skin of 27 volunteers. The average value (± SD) of mitoPO₂ was 44 ± 17 mmHg and mean mitoVO₂ values were 5.8 ± 2.3 and 6.1 ± 1.6 mmHg s^-1 at a skin temperature of 34°C and 40°C, respectively. No major discomfort during measurement and no long-term dermatological abnormalities were reported in a survey performed 1 month after measurements.ConclusionThese results show that the clinical prototype allows measurement of mitochondrial oxygenation and oxygen consumption in humans. The development of this clinically applicable device offers opportunities for further evaluation of the technique in humans and the start of first clinical studies.     

In vivo brain imaging of mitochondrial Ca2+ in neurodegenerative diseases with multiphoton microscopy

Mitochondria are involved in a large number of essential roles related to neuronal function. Ca²⁺ handling by mitochondria is critical for many of these functions, including energy production and cellular fate. Conversely, mitochondrial Ca²⁺ mishandling has been related to a variety of neurodegenerative diseases. Investigating mitochondrial Ca²⁺ dynamics is essential for advancing our understanding of the role of intracellular mitochondrial Ca²⁺ signals in physiology and pathology. Improved Ca²⁺ indicators, and the ability to target them to different cells and compartments, have emerged as useful tools for analysis of Ca²⁺ signals in living organisms. Combined with state-of-the-art techniques such as multiphoton microscopy, they allow for the study of mitochondrial Ca²⁺ dynamics in vivo in mouse models of the disease. Here, we provide an overview of the Ca²⁺ transporters/ion channels in mitochondrial membranes, and the involvement of mitochondrial Ca²⁺ in neurodegenerative diseases followed by a summary of the main tools available to evaluate mitochondrial Ca²⁺ dynamics in vivo using the aforementioned technique.