Angela Logan

Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS

Ischaemia-reperfusion injury occurs when the blood supply to an organ is disrupted and then restored, and underlies many disorders, notably heart attack and stroke. While reperfusion of ischaemic tissue is essential for survival, it also initiates oxidative damage, cell death and aberrant immune responses through the generation of mitochondrial reactive oxygen species (ROS). Although mitochondrial ROS production in ischaemia reperfusion is established, it has generally been considered a nonspecific response to reperfusion. Here we develop a comparative in vivo metabolomic analysis, and unexpectedly identify widely conserved metabolic pathways responsible for mitochondrial ROS production during ischaemia reperfusion. We show that selective accumulation of the citric acid cycle intermediate succinate is a universal metabolic signature of ischaemia in a range of tissues and is responsible for mitochondrial ROS production during reperfusion. Ischaemic succinate accumulation arises from reversal of succinate dehydrogenase, which in turn is driven by fumarate overflow from purine nucleotide breakdown and partial reversal of the malate/aspartate shuttle. After reperfusion, the accumulated succinate is rapidly re-oxidized by succinate dehydrogenase, driving extensive ROS generation by reverse electron transport at mitochondrial complex I. Decreasing ischaemic succinate accumulation by pharmacological inhibition is sufficient to ameliorate in vivo ischaemia-reperfusion injury in murine models of heart attack and stroke. Thus, we have identified a conserved metabolic response of tissues to ischaemia and reperfusion that unifies many hitherto unconnected aspects of ischaemia-reperfusion injury. Furthermore, these findings reveal a new pathway for metabolic control of ROS production in vivo, while demonstrating that inhibition of ischaemic succinate accumulation and its oxidation after subsequent reperfusion is a potential therapeutic target to decrease ischaemia-reperfusion injury in a range of pathologies.

Cardioprotection by S-nitrosation of a cysteine switch on mitochondrial complex I

Oxidative damage from elevated production of reactive oxygen species (ROS) contributes to ischemia-reperfusion injury in myocardial infarction and stroke. The mechanism by which the increase in ROS occurs is not known, and it is unclear how this increase can be prevented. A wide variety of nitric oxide donors and S-nitrosating agents protect the ischemic myocardium from infarction, but the responsible mechanisms are unclear. Here we used a mitochondria-selective S-nitrosating agent, MitoSNO, to determine how mitochondrial S-nitrosation at the reperfusion phase of myocardial infarction is cardioprotective in vivo in mice. We found that protection is due to the S-nitrosation of mitochondrial complex I, which is the entry point for electrons from NADH into the respiratory chain. Reversible S-nitrosation of complex I slows the reactivation of mitochondria during the crucial first minutes of the reperfusion of ischemic tissue, thereby decreasing ROS production, oxidative damage and tissue necrosis. Inhibition of complex I is afforded by the selective S-nitrosation of Cys39 on the ND3 subunit, which becomes susceptible to modification only after ischemia. Our results identify rapid complex I reactivation as a central pathological feature of ischemia-reperfusion injury and show that preventing this reactivation by modification of a cysteine switch is a robust cardioprotective mechanism and hence a rational therapeutic strategy.

Measurement of H2O2 within Living Drosophila during Aging Using a Ratiometric Mass Spectrometry Probe Targeted to the Mitochondrial Matrix

Summary Hydrogen peroxide (H2O2) is central to mitochondrial oxidative damage and redox signaling, but its roles are poorly understood due to the difficulty of measuring mitochondrial H2O2 in vivo. Here we report a ratiometric mass spectrometry probe approach to assess mitochondrial matrix H2O2 levels in vivo. The probe, MitoB, comprises a triphenylphosphonium (TPP) cation driving its accumulation within mitochondria, conjugated to an arylboronic acid that reacts with H2O2 to form a phenol, MitoP. Quantifying the MitoP/MitoB ratio by liquid chromatography-tandem mass spectrometry enabled measurement of a weighted average of mitochondrial H2O2 that predominantly reports on thoracic muscle mitochondria within living flies. There was an increase in mitochondrial H2O2 with age in flies, which was not coordinately altered by interventions that modulated life span. Our findings provide approaches to investigate mitochondrial ROS in vivo and suggest that while an increase in overall mitochondrial H2O2 correlates with aging, it may not be causative.

DNA Damage Links Mitochondrial Dysfunction to Atherosclerosis and the Metabolic Syndrome

Rationale: DNA damage is present in both genomic and mitochondrial DNA in atherosclerosis. However, whether DNA damage itself promotes atherosclerosis, or is simply a byproduct of the risk factors that promote atherosclerosis, is unknown. Objective: To examine the effect of DNA damage on atherosclerosis, we studied apolipoprotein (Apo)E−/− mice that were haploinsufficient for the protein kinase ATM (ataxia telangiectasia mutated), which coordinates DNA repair. Methods and Results: ATM+/−/ApoE−/− mice developed accelerated atherosclerosis and multiple features of the metabolic syndrome, including hypertension, hypercholesterolemia, obesity, steatohepatitis, and glucose intolerance. Transplantation with ATM+/+ bone marrow attenuated atherosclerosis but not the metabolic syndrome. ATM+/− smooth muscle cells and macrophages showed increased nuclear DNA damage and defective DNA repair signaling, growth arrest, and apoptosis. Metabolomic screening of ATM+/−/ApoE−/− mouse tissues identified metabolic changes compatible with mitochondrial defects, with increased &bgr;-hydroxybutyrate but reduced lactate, reduced glucose, and alterations in multiple lipid species. ATM+/−/ApoE−/− mouse tissues showed an increased frequency of a mouse mitochondrial “common” deletion equivalent and reduced mitochondrial oxidative phosphorylation. Conclusions: We propose that failure of DNA repair generates defects in cell proliferation, apoptosis, and mitochondrial dysfunction. This in turn leads to ketosis, hyperlipidemia, and increased fat storage, promoting atherosclerosis and the metabolic syndrome. Prevention of mitochondrial dysfunction may represent a novel target in cardiovascular disease.

Consequences of long-term oral administration of the mitochondria-targeted antioxidant MitoQ to wild-type mice.

The mitochondria-targeted quinone MitoQ protects mitochondria in animal studies of pathologies in vivo and is being developed as a therapy for humans. However, it is unclear whether the protective action of MitoQ is entirely due to its antioxidant properties, because long-term MitoQ administration may alter whole-body metabolism and gene expression. To address this point, we administered high levels of MitoQ orally to wild-type C57BL/6 mice for up to 28 weeks and investigated the effects on whole-body physiology, metabolism, and gene expression, finding no measurable deleterious effects. In addition, because antioxidants can act as pro-oxidants under certain conditions in vitro, we examined the effects of MitoQ administration on markers of oxidative damage. There were no changes in the expression of mitochondrial or antioxidant genes as assessed by DNA microarray analysis. There were also no increases in oxidative damage to mitochondrial protein, DNA, or cardiolipin, and the activities of mitochondrial enzymes were unchanged. Therefore, MitoQ does not act as a pro-oxidant in vivo. These findings indicate that mitochondria-targeted antioxidants can be safely administered long-term to wild-type mice.

Mitochondrial DNA Damage Can Promote Atherosclerosis Independently of Reactive Oxygen Species Through Effects on Smooth Muscle Cells and Monocytes and Correlates With Higher-Risk Plaques in Humans

Background— Mitochondrial DNA (mtDNA) damage occurs in both circulating cells and the vessel wall in human atherosclerosis. However, it is unclear whether mtDNA damage directly promotes atherogenesis or is a consequence of tissue damage, which cell types are involved, and whether its effects are mediated only through reactive oxygen species. Methods and Results— mtDNA damage occurred early in the vessel wall in apolipoprotein E–null (ApoE−/−) mice, before significant atherosclerosis developed. mtDNA defects were also identified in circulating monocytes and liver and were associated with mitochondrial dysfunction. To determine whether mtDNA damage directly promotes atherosclerosis, we studied ApoE−/− mice deficient for mitochondrial polymerase-&ggr; proofreading activity (polG−/−/ApoE−/−). polG−/−/ApoE−/− mice showed extensive mtDNA damage and defects in oxidative phosphorylation but no increase in reactive oxygen species. polG−/−/ApoE−/− mice showed increased atherosclerosis, associated with impaired proliferation and apoptosis of vascular smooth muscle cells, and hyperlipidemia. Transplantation with polG−/−/ApoE−/− bone marrow increased the features of plaque vulnerability, and polG−/−/ApoE−/− monocytes showed increased apoptosis and inflammatory cytokine release. To examine mtDNA damage in human atherosclerosis, we assessed mtDNA adducts in plaques and in leukocytes from patients who had undergone virtual histology intravascular ultrasound characterization of coronary plaques. Human atherosclerotic plaques showed increased mtDNA damage compared with normal vessels; in contrast, leukocyte mtDNA damage was associated with higher-risk plaques but not plaque burden. Conclusions— We show that mtDNA damage in vessel wall and circulating cells is widespread and causative and indicates higher risk in atherosclerosis. Protection against mtDNA damage and improvement of mitochondrial function are potential areas for new therapeutics.

Mitochondrial and nuclear DNA matching shapes metabolism and healthy ageing

Human mitochondrial DNA (mtDNA) shows extensive within-population sequence variability. Many studies suggest that mtDNA variants may be associated with ageing or diseases, although mechanistic evidence at the molecular level is lacking. Mitochondrial replacement has the potential to prevent transmission of disease-causing oocyte mtDNA. However, extension of this technology requires a comprehensive understanding of the physiological relevance of mtDNA sequence variability and its match with the nuclear-encoded mitochondrial genes. Studies in conplastic animals allow comparison of individuals with the same nuclear genome but different mtDNA variants, and have provided both supporting and refuting evidence that mtDNA variation influences organismal physiology. However, most of these studies did not confirm the conplastic status, focused on younger animals, and did not investigate the full range of physiological and phenotypic variability likely to be influenced by mitochondria. Here we systematically characterized conplastic mice throughout their lifespan using transcriptomic, proteomic, metabolomic, biochemical, physiological and phenotyping studies. We show that mtDNA haplotype profoundly influences mitochondrial proteostasis and reactive oxygen species generation, insulin signalling, obesity, and ageing parameters including telomere shortening and mitochondrial dysfunction, resulting in profound differences in health longevity between conplastic strains.

Mitochondria‐Targeted Antioxidants in the Treatment of Disease

Mitochondrial oxidative damage is thought to contribute to a wide range of human diseases; therefore, the development of approaches to decrease this damage may have therapeutic potential. Mitochondria‐targeted antioxidants that selectively block mitochondrial oxidative damage and prevent some types of cell death have been developed. These compounds contain antioxidant moieties, such as ubiquinone, tocopherol, or nitroxide, that are targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation. Because of the large mitochondrial membrane potential, the cations are accumulated within the mitochondria inside cells. There, the conjugated antioxidant moiety protects mitochondria from oxidative damage. Here, we outline some of the work done to date on these compounds and how they may be developed as therapies.

Rapid uptake of lipophilic triphenylphosphonium cations by mitochondria in vivo following intravenous injection: Implications for mitochondria-specific therapies and probes

BACKGROUND Mitochondrial dysfunction contributes to a range of pathologies, consequently there is a need to monitor mitochondrial function and to intervene pharmacologically to prevent mitochondrial damage. One approach to this is to deliver antioxidants, probes and pharmacophores to mitochondria by conjugation to the lipophilic triphenylphosphonium (TPP) cation that is taken up selectively by mitochondria driven by the membrane potential. CONCLUSIONS Oral administration of TPP-conjugated antioxidants protects against mitochondrial damage in vivo. However, there is also a need to deliver molecules rapidly to mitochondria to respond quickly to pathologies and for the real-time assessment of mitochondrial function. METHODS To see if this was possible we investigated how rapidly TPP cations were taken up by mitochondria in vivo following intravenous (iv) administration. RESULTS AlkylTPP cations were accumulated selectively by mitochondria within mice within 5 min of iv injection. The extent of uptake was enhanced 10-30-fold relative to simple alkylTPP cations by attaching functional groups to the TPP cation via long, hydrophobic alkyl chains. Conclusions: Mitochondria-targeted antioxidants, probes and pharmacophores can be delivered into mitochondria within minutes of iv administration. GENERAL SIGNIFICANCE These findings greatly extend the utility of mitochondria-targeted lipophilic cations as therapies and probes.

Antioxidant properties of MitoTEMPOL and its hydroxylamine

Piperidine nitroxides such as TEMPOL have been widely used as antioxidants in vitro and in vivo. MitoTEMPOL is a mitochondria-targeted derivative of TEMPOL designed to protect mitochondria from the oxidative damage that they accumulate, but once there is rapidly reduced to its hydroxylamine, MitoTEMPOL-H. As little is known about the antioxidant efficacy of hydroxylamines, this study has assessed the antioxidant activity of both MitoTEMPOL and MitoTEMPOL-H. The hydroxylamine was more effective at preventing lipid-peroxidation than MitoTEMPOL and decreased oxidative damage to mitochondrial DNA caused by menadione. In contrast to MitoTEMPOL, MitoTEMPOL-H has no superoxide dismutase activity and its antioxidant actions are likely to be mediated by hydrogen atom donation. Therefore, even though MitoTEMPOL is rapidly reduced to MitoTEMPOL-H in cells, it remains an effective antioxidant. Furthermore, as TEMPOL is also reduced to a hydroxylamine in vivo, many of its antioxidant effects may also be mediated by its hydroxylamine.