Publications by authors named "Richard C Hartley"

72 Publications

Targeting Methylglyoxal in Diabetic Kidney Disease Using the Mitochondria-Targeted Compound MitoGamide.

Nutrients 2021 Apr 25;13(5). Epub 2021 Apr 25.

Department of Diabetes, Central Clinical, School, Alfred Medical Research and Education Precinct, Monash University, Melbourne, VIC 3004, Australia.

Diabetic kidney disease (DKD) remains the number one cause of end-stage renal disease in the western world. In experimental diabetes, mitochondrial dysfunction in the kidney precedes the development of DKD. Reactive 1,2-dicarbonyl compounds, such as methylglyoxal, are generated from sugars both endogenously during diabetes and exogenously during food processing. Methylglyoxal is thought to impair the mitochondrial function and may contribute to the pathogenesis of DKD. Here, we sought to target methylglyoxal within the mitochondria using MitoGamide, a mitochondria-targeted dicarbonyl scavenger, in an experimental model of diabetes. Male 6-week-old heterozygous Akita mice (C57BL/6-Ins2-Akita/J) or wildtype littermates were randomized to receive MitoGamide (10 mg/kg/day) or a vehicle by oral gavage for 16 weeks. MitoGamide did not alter the blood glucose control or body composition. Akita mice exhibited hallmarks of DKD including albuminuria, hyperfiltration, glomerulosclerosis, and renal fibrosis, however, after 16 weeks of treatment, MitoGamide did not substantially improve the renal phenotype. Complex-I-linked mitochondrial respiration was increased in the kidney of Akita mice which was unaffected by MitoGamide. Exploratory studies using transcriptomics identified that MitoGamide induced changes to olfactory signaling, immune system, respiratory electron transport, and post-translational protein modification pathways. These findings indicate that targeting methylglyoxal within the mitochondria using MitoGamide is not a valid therapeutic approach for DKD and that other mitochondrial targets or processes upstream should be the focus of therapy.
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http://dx.doi.org/10.3390/nu13051457DOI Listing
April 2021

Photoactivated release of membrane impermeant sulfonates inside cells.

Chem Commun (Camb) 2021 Apr 23;57(32):3917-3920. Epub 2021 Mar 23.

School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.

Photouncaging delivers compounds with high spatial and temporal control to induce or inhibit biological processes but the released compounds may diffuse out. We here demonstrate that sulfonate anions can be photocaged so that a membrane impermeable compound can enter cells, be uncaged by photoirradiation and trapped within the cell.
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http://dx.doi.org/10.1039/d0cc07713eDOI Listing
April 2021

Tetra-arylborate lipophilic anions as targeting groups.

Chem Commun (Camb) 2021 Mar 26;57(25):3147-3150. Epub 2021 Feb 26.

School of Chemistry, University of Glasgow, Glasgow, G12 8QQ, UK.

Tetraphenylborate (TPB) anions traverse membranes but are excluded from mitochondria by the membrane potential (Δψ). TPB-conjugates also distributed across membranes in response to Δψ, but surprisingly, they rapidly entered cells. They accumulated within lysosomes following endocystosis. This pH-independent targeting of lysosomes makes possible new classes of probe and bioactive molecules.
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http://dx.doi.org/10.1039/d0cc07924cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8062962PMC
March 2021

Nrf2 is activated by disruption of mitochondrial thiol homeostasis but not by enhanced mitochondrial superoxide production.

J Biol Chem 2020 Dec 9. Epub 2020 Dec 9.

University of Cambridge, United Kingdom.

The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates the expression of genes involved in antioxidant defenses to modulate fundamental cellular processes such as mitochondrial function and glutathione metabolism. Previous reports proposed that mitochondrial ROS production and disruption of the glutathione pool activate the Nrf2 pathway, suggesting that Nrf2 senses mitochondrial redox signals and/or oxidative damage and signals to the nucleus to respond appropriately. However, until now it has not been possible to disentangle the overlapping effects of mitochondrial superoxide/ hydrogen peroxide production as a redox signal from changes to mitochondrial thiol homeostasis on Nrf2. Recently, we developed mitochondria-targeted reagents that can independently induce mitochondrial superoxide and hydrogen peroxide production (MitoPQ), or selectively disrupt mitochondrial thiol homeostasis (MitoCDNB). Using these reagents, here we have determined how enhanced generation of mitochondrial superoxide and hydrogen peroxide, or disruption of mitochondrial thiol homeostasis affect activation of the Nrf2 system in cells, which was assessed by Nrf2 protein level, nuclear translocation and expression of its target genes. We found that selective disruption of the mitochondrial glutathione pool and inhibition of its thioredoxin system by MitoCDNB led to Nrf2 activation, while using MitoPQ to enhance production of mitochondrial superoxide and hydrogen peroxide alone did not. We further showed that Nrf2 activation by MitoCDNB requires cysteine sensors of Kelch-like ECH-associated protein 1 (Keap1). These findings provide important information on how disruption to mitochondrial redox homeostasis is sensed in the cytoplasm and signaled to the nucleus.
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http://dx.doi.org/10.1074/jbc.RA120.016551DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7948991PMC
December 2020

Enhancing the Mitochondrial Uptake of Phosphonium Cations by Carboxylic Acid Incorporation.

Front Chem 2020 9;8:783. Epub 2020 Sep 9.

School of Chemistry, University of Glasgow, Glasgow, United Kingdom.

There is considerable interest in developing drugs and probes targeted to mitochondria in order to understand and treat the many pathologies associated with mitochondrial dysfunction. The large membrane potential, negative inside, across the mitochondrial inner membrane enables delivery of molecules conjugated to lipophilic phosphonium cations to the organelle. Due to their combination of charge and hydrophobicity, quaternary triarylphosphonium cations rapidly cross biological membranes without the requirement for a carrier. Their extent of uptake is determined by the magnitude of the mitochondrial membrane potential, as described by the Nernst equation. To further enhance this uptake here we explored whether incorporation of a carboxylic acid into a quaternary triarylphosphonium cation would enhance its mitochondrial uptake in response to both the membrane potential and the mitochondrial pH gradient (alkaline inside). Accumulation of arylpropionic acid derivatives depended on both the membrane potential and the pH gradient. However, acetic or benzoic derivatives did not accumulate, due to their lowered pK. Surprisingly, despite not being taken up by mitochondria, the phenylacetic or phenylbenzoic derivatives were not retained within mitochondria when generated within the mitochondrial matrix by hydrolysis of their cognate esters. Computational studies, supported by crystallography, showed that these molecules passed through the hydrophobic core of mitochondrial inner membrane as a neutral dimer. This finding extends our understanding of the mechanisms of membrane permeation of lipophilic cations and suggests future strategies to enhance drug and probe delivery to mitochondria.
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http://dx.doi.org/10.3389/fchem.2020.00783DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7509049PMC
September 2020

Confirmation of the Cardioprotective Effect of MitoGamide in the Diabetic Heart.

Cardiovasc Drugs Ther 2020 12 26;34(6):823-834. Epub 2020 Sep 26.

Department of Medicine, University of Cambridge, Cambridge, UK.

Purpose: HFpEF (heart failure with preserved ejection fraction) is a major consequence of diabetic cardiomyopathy with no effective treatments. Here, we have characterized Akita mice as a preclinical model of HFpEF and used it to confirm the therapeutic efficacy of the mitochondria-targeted dicarbonyl scavenger, MitoGamide.

Methods And Results: A longitudinal echocardiographic analysis confirmed that Akita mice develop diastolic dysfunction with reduced E peak velocity, E/A ratio and extended isovolumetric relaxation time (IVRT), while the systolic function remains comparable with wild-type mice. The myocardium of Akita mice had a decreased ATP/ADP ratio, elevated mitochondrial oxidative stress and increased organelle density, compared with that of wild-type mice. MitoGamide, a mitochondria-targeted 1,2-dicarbonyl scavenger, exhibited good stability in vivo, uptake into cells and mitochondria and reactivity with dicarbonyls. Treatment of Akita mice with MitoGamide for 12 weeks significantly improved the E/A ratio compared with the vehicle-treated group.

Conclusion: Our work confirms that the Akita mouse model of diabetes replicates key clinical features of diabetic HFpEF, including cardiac and mitochondrial dysfunction. Furthermore, in this independent study, MitoGamide treatment improved diastolic function in Akita mice.
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http://dx.doi.org/10.1007/s10557-020-07086-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7674384PMC
December 2020

Targeting succinate dehydrogenase with malonate ester prodrugs decreases renal ischemia reperfusion injury.

Redox Biol 2020 09 12;36:101640. Epub 2020 Jul 12.

Department of Surgery and Cambridge NIHR Biomedical Research Centre, Biomedical Campus, University of Cambridge, Cambridge, CB2 0QQ, UK. Electronic address:

Renal ischemia reperfusion (IR) injury leads to significant patient morbidity and mortality, and its amelioration is an urgent unmet clinical need. Succinate accumulates during ischemia and its oxidation by the mitochondrial enzyme succinate dehydrogenase (SDH) drives the ROS production that underlies IR injury. Consequently, compounds that inhibit SDH may have therapeutic potential against renal IR injury. Among these, the competitive SDH inhibitor malonate, administered as a cell-permeable malonate ester prodrug, has shown promise in models of cardiac IR injury, but the efficacy of malonate ester prodrugs against renal IR injury have not been investigated. Here we show that succinate accumulates during ischemia in mouse, pig and human models of renal IR injury, and that its rapid oxidation by SDH upon reperfusion drives IR injury. We then show that the malonate ester prodrug, dimethyl malonate (DMM), can ameliorate renal IR injury when administered at reperfusion but not prior to ischemia in the mouse. Finally, we show that another malonate ester prodrug, diacetoxymethyl malonate (MAM), is more potent than DMM because of its faster esterase hydrolysis. Our data show that the mitochondrial mechanisms of renal IR injury are conserved in the mouse, pig and human and that inhibition of SDH by 'tuned' malonate ester prodrugs, such as MAM, is a promising therapeutic strategy in the treatment of clinical renal IR injury.
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http://dx.doi.org/10.1016/j.redox.2020.101640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7372157PMC
September 2020

Selective Delivery of Dicarboxylates to Mitochondria by Conjugation to a Lipophilic Cation via a Cleavable Linker.

Mol Pharm 2020 09 5;17(9):3526-3540. Epub 2020 Aug 5.

Molecular Research Center, Mitochondrial Biology Unit, Biomedical Campus, University of Cambridge, Cambridge CB2 0XY, United Kingdom.

Many mitochondrial metabolites and bioactive molecules contain two carboxylic acid moieties that make them unable to cross biological membranes. Hence, there is considerable interest in facilitating the uptake of these molecules into cells and mitochondria to modify or report on their function. Conjugation to the triphenylphosphonium (TPP) lipophilic cation is widely used to deliver molecules selectively to mitochondria in response to the membrane potential. However, permanent attachment to the cation can disrupt the biological function of small dicarboxylates. Here, we have developed a strategy using TPP to release dicarboxylates selectively within mitochondria. For this, the dicarboxylate is attached to a TPP compound via a single ester bond, which is then cleaved by intramitochondrial esterase activity, releasing the dicarboxylate within the organelle. Leaving the second carboxylic acid free also means mitochondrial uptake is dependent on the pH gradient across the inner membrane. To assess this strategy, we synthesized a range of TPP monoesters of the model dicarboxylate, malonate. We then tested their mitochondrial accumulation and ability to deliver malonate to isolated mitochondria and to cells, and . A TPP-malonate monoester compound, TPP-malonate, in which the dicarboxylate group was attached to the TPP compound via a hydrophobic undecyl link, was most effective at releasing malonate within mitochondria in cells and . Therefore, we have developed a TPP-monoester platform that enables the selective release of bioactive dicarboxylates within mitochondria.
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http://dx.doi.org/10.1021/acs.molpharmaceut.0c00533DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7482397PMC
September 2020

Ester Prodrugs of Malonate with Enhanced Intracellular Delivery Protect Against Cardiac Ischemia-Reperfusion Injury In Vivo.

Cardiovasc Drugs Ther 2020 Jul 9. Epub 2020 Jul 9.

Department of Medicine, University of Cambridge, Cambridge, CB2 0QQ, UK.

Purpose: Mitochondrial reactive oxygen species (ROS) production upon reperfusion of ischemic tissue initiates the ischemia/reperfusion (I/R) injury associated with heart attack. During ischemia, succinate accumulates and its oxidation upon reperfusion by succinate dehydrogenase (SDH) drives ROS production. Inhibition of succinate accumulation and/or oxidation by dimethyl malonate (DMM), a cell permeable prodrug of the SDH inhibitor malonate, can decrease I/R injury. However, DMM is hydrolysed slowly, requiring administration to the heart prior to ischemia, precluding its administration to patients at the point of reperfusion, for example at the same time as unblocking a coronary artery following a heart attack. To accelerate malonate delivery, here we developed more rapidly hydrolysable malonate esters.

Methods: We synthesised a series of malonate esters and assessed their uptake and hydrolysis by isolated mitochondria, C2C12 cells and in mice in vivo. In addition, we assessed protection against cardiac I/R injury by the esters using an in vivo mouse model of acute myocardial infarction.

Results: We found that the diacetoxymethyl malonate diester (MAM) most rapidly delivered large amounts of malonate to cells in vivo. Furthermore, MAM could inhibit mitochondrial ROS production from succinate oxidation and was protective against I/R injury in vivo when added at reperfusion.

Conclusions: The rapidly hydrolysed malonate prodrug MAM can protect against cardiac I/R injury in a clinically relevant mouse model.
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http://dx.doi.org/10.1007/s10557-020-07033-6DOI Listing
July 2020

Mitochondria-targeted paraquat and metformin mediate ROS production to induce multiple pathways of retrograde signaling: A dose-dependent phenomenon.

Redox Biol 2020 09 21;36:101606. Epub 2020 Jun 21.

Department of Biomedical Sciences, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA. Electronic address:

The mitochondrial electron transport chain is a major source of reactive oxygen species (ROS) and is also a target of ROS, with an implied role in the stabilization of hypoxia-inducible factor (HIF) and induction of the AMPK pathway. Here we used varying doses of two agents, Mito-Paraquat and Mito-Metformin, that have been conjugated to cationic triphenylphosphonium (TPP) moiety to selectively target them to the mitochondrial matrix compartment, thereby resulting in the site-specific generation of ROS within mitochondria. These agents primarily induce superoxide (O) production by acting on complex I. In Raw264.7 macrophages, C2C12 skeletal myocytes, and HCT116 adenocarcinoma cells, we show that mitochondria-targeted oxidants can induce ROS (O and HO). In all three cell lines tested, the mitochondria-targeted agents disrupted membrane potential and activated calcineurin and the Cn-dependent retrograde signaling pathway. Hypoxic culture conditions also induced Cn activation and HIF1α activation in a temporally regulated manner, with the former appearing at shorter exposure times. Together, our results indicate that mitochondrial oxidant-induced retrograde signaling is driven by disruption of membrane potential and activation of Ca/Cn pathway and is independent of ROS-induced HIF1α or AMPK pathways.
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http://dx.doi.org/10.1016/j.redox.2020.101606DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327929PMC
September 2020

Succinate accumulation drives ischaemia-reperfusion injury during organ transplantation.

Nat Metab 2019 09;1:966-974

MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge, UK.

During heart transplantation, storage in cold preservation solution is thought to protect the organ by slowing metabolism; by providing osmotic support; and by minimising ischaemia-reperfusion (IR) injury upon transplantation into the recipient. Despite its widespread use our understanding of the metabolic changes prevented by cold storage and how warm ischaemia leads to damage is surprisingly poor. Here, we compare the metabolic changes during warm ischaemia (WI) and cold ischaemia (CI) in hearts from mouse, pig, and human. We identify common metabolic alterations during WI and those affected by CI, thereby elucidating mechanisms underlying the benefits of CI, and how WI causes damage. Succinate accumulation is a major feature within ischaemic hearts across species, and CI slows succinate generation, thereby reducing tissue damage upon reperfusion caused by the production of mitochondrial reactive oxygen species (ROS). Importantly, the inevitable periods of WI during organ procurement lead to the accumulation of damaging levels of succinate during transplantation, despite cooling organs as rapidly as possible. This damage is ameliorated by metabolic inhibitors that prevent succinate accumulation and oxidation. Our findings suggest how WI and CI contribute to transplant outcome and indicate new therapies for improving the quality of transplanted organs.
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http://dx.doi.org/10.1038/s42255-019-0115-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7212038PMC
September 2019

Correction to: The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart.

Cardiovasc Drugs Ther 2020 Apr;34(2):223

Heart Failure Pharmacology, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

The article "The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart".
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http://dx.doi.org/10.1007/s10557-019-06929-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7645541PMC
April 2020

A sensitive mass spectrometric assay for mitochondrial CoQ pool redox state in vivo.

Free Radic Biol Med 2020 02 5;147:37-47. Epub 2019 Dec 5.

MRC Mitochondrial Biology Unit, University of Cambridge, Hills Road, Cambridge, CB2 0XY, UK; Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 0QQ, UK. Electronic address:

Coenzyme Q (CoQ) is an essential cofactor, primarily found in the mitochondrial inner membrane where it functions as an electron carrier in the respiratory chain, and as a lipophilic antioxidant. The redox state of the CoQ pool is the ratio of its oxidised (ubiquinone) and reduced (ubiquinol) forms, and is a key indicator of mitochondrial bioenergetic and antioxidant status. However, the role of CoQ redox state in vivo is poorly understood, because determining its value is technically challenging due to redox changes during isolation, extraction and analysis. To address these problems, we have developed a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay that enables us to extract and analyse both the CoQ redox state and the magnitude of the CoQ pool with negligible changes to redox state from small amounts of tissue. This will enable the physiological and pathophysiological roles of the CoQ redox state to be investigated in vivo.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.11.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6975167PMC
February 2020

The Mitochondria-Targeted Methylglyoxal Sequestering Compound, MitoGamide, Is Cardioprotective in the Diabetic Heart.

Cardiovasc Drugs Ther 2019 12;33(6):669-674

Heart Failure Pharmacology, Baker Heart and Diabetes Institute, Melbourne, VIC, Australia.

Purpose: Methylglyoxal, a by-product of glycolysis and a precursor in the formation of advanced glycation end-products, is significantly elevated in the diabetic myocardium. Therefore, we sought to investigate the mitochondria-targeted methylglyoxal scavenger, MitoGamide, in an experimental model of spontaneous diabetic cardiomyopathy.

Methods: Male 6-week-old Akita or wild type mice received daily oral gavage of MitoGamide or vehicle for 10 weeks. Several morphological and systemic parameters were assessed, as well as cardiac function by echocardiography.

Results: Akita mice were smaller in size than wild type counterparts in terms of body weight and tibial length. Akita mice exhibited elevated blood glucose and glycated haemoglobin. Total heart and individual ventricles were all smaller in Akita mice. None of the aforementioned parameters was impacted by MitoGamide treatment. Echocardiographic analysis confirmed that cardiac dimensions were smaller in Akita hearts. Diastolic dysfunction was evident in Akita mice, and notably, MitoGamide treatment preferentially improved several of these markers, including e'/a' ratio and E/e' ratio.

Conclusions: Our findings suggest that MitoGamide, a novel mitochondria-targeted approach, offers cardioprotection in experimental diabetes and therefore may offer therapeutic potential for the treatment of cardiomyopathy in patients with diabetes.
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http://dx.doi.org/10.1007/s10557-019-06914-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994445PMC
December 2019

Nrf2 controls iron homeostasis in haemochromatosis and thalassaemia via Bmp6 and hepcidin.

Nat Metab 2019 05 13;1(5):519-531. Epub 2019 May 13.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, OX3 9DS, UK.

Iron is critical for life but toxic in excess because of iron-catalysed formation of pro-oxidants that cause tissue damage in a range of disorders. The Nrf2 transcription factor orchestrates cell-intrinsic protective antioxidant responses, and the peptide hormone hepcidin maintains systemic iron homeostasis, but is pathophysiologically decreased in haemochromatosis and beta-thalassaemia. Here, we show that Nrf2 is activated by iron-induced, mitochondria-derived pro-oxidants and drives Bmp6 expression in liver sinusoid endothelial cells, which in turn increases hepcidin synthesis by neighbouring hepatocytes. In Nrf2 knockout mice, the Bmp6-hepcidin response to oral and parenteral iron is impaired and iron accumulation and hepatic damage are increased. Pharmacological activation of Nrf2 stimulates the Bmp6-hepcidin axis, improving iron homeostasis in haemochromatosis and counteracting the inhibition of Bmp6 by erythroferrone in beta-thalassaemia. We propose that Nrf2 links cellular sensing of excess toxic iron to control of systemic iron homeostasis and antioxidant responses, and may be a therapeutic target for iron-associated disorders.
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http://dx.doi.org/10.1038/s42255-019-0063-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6609153PMC
May 2019

Detection of changes in mitochondrial hydrogen sulfide in the fish model (Poeciliidae).

Biol Open 2019 May 9;8(5). Epub 2019 May 9.

MRC Mitochondrial Biology Unit, University of Cambridge, Hills Road, Cambridge CB2 0XY, UK

In this paper, we outline the use of a mitochondria-targeted ratiometric mass spectrometry probe, MitoA, to detect changes in mitochondrial hydrogen sulfide (HS) in (family Poeciliidae). MitoA is introduced via intraperitoneal injection into the animal and is taken up by mitochondria, where it reacts with HS to form the product MitoN. The MitoN/MitoA ratio can be used to assess relative changes in the amounts of mitochondrial HS produced over time. We describe the use of MitoA in the fish species to illustrate the steps for adopting the use of MitoA in a new organism, including extraction and purification of MitoA and MitoN from tissues followed by tandem mass spectrometry. In this proof-of-concept study we exposed HS tolerant to 59 µM free HS for 5 h, which resulted in increased MitoN/MitoA in brain and gills, but not in liver or muscle, demonstrating increased mitochondrial HS levels in select tissues following whole-animal HS exposure. This is the first time that accumulation of HS has been observed during whole-animal exposure to free HS using MitoA. This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/bio.041467DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6550084PMC
May 2019

Selective mitochondrial superoxide generation in vivo is cardioprotective through hormesis.

Free Radic Biol Med 2019 04 4;134:678-687. Epub 2019 Feb 4.

Department of Medicine, University of Cambridge, Hills Road, Cambridge, CB2 0XY, UK. Electronic address:

Reactive oxygen species (ROS) have an equivocal role in myocardial ischaemia reperfusion injury. Within the cardiomyocyte, mitochondria are both a major source and target of ROS. We evaluate the effects of a selective, dose-dependent increase in mitochondrial ROS levels on cardiac physiology using the mitochondria-targeted redox cycler MitoParaquat (MitoPQ). Low levels of ROS decrease the susceptibility of neonatal rat ventricular myocytes (NRVMs) to anoxia/reoxygenation injury and also cause profound protection in an in vivo mouse model of ischaemia/reperfusion. However higher doses of MitoPQ resulted in a progressive alteration of intracellular [Ca] homeostasis and mitochondrial function in vitro, leading to dysfunction and death at high doses. Our data show that a primary increase in mitochondrial ROS can alter cellular function, and support a hormetic model in which low levels of ROS are cardioprotective while higher levels of ROS are cardiotoxic.
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http://dx.doi.org/10.1016/j.freeradbiomed.2019.01.034DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6607027PMC
April 2019

Selective Disruption of Mitochondrial Thiol Redox State in Cells and In Vivo.

Cell Chem Biol 2019 03 31;26(3):449-461.e8. Epub 2019 Jan 31.

MRC Mitochondrial Biology Unit, University of Cambridge, Cambridge Biomedical Campus, Cambridge CB2 0XY, UK; Department of Medicine, University of Cambridge, Cambridge CB2 0QQ, UK. Electronic address:

Mitochondrial glutathione (GSH) and thioredoxin (Trx) systems function independently of the rest of the cell. While maintenance of mitochondrial thiol redox state is thought vital for cell survival, this was not testable due to the difficulty of manipulating the organelle's thiol systems independently of those in other cell compartments. To overcome this constraint we modified the glutathione S-transferase substrate and Trx reductase (TrxR) inhibitor, 1-chloro-2,4-dinitrobenzene (CDNB) by conjugation to the mitochondria-targeting triphenylphosphonium cation. The result, MitoCDNB, is taken up by mitochondria where it selectively depletes the mitochondrial GSH pool, catalyzed by glutathione S-transferases, and directly inhibits mitochondrial TrxR2 and peroxiredoxin 3, a peroxidase. Importantly, MitoCDNB inactivates mitochondrial thiol redox homeostasis in isolated cells and in vivo, without affecting that of the cytosol. Consequently, MitoCDNB enables assessment of the biomedical importance of mitochondrial thiol homeostasis in reactive oxygen species production, organelle dynamics, redox signaling, and cell death in cells and in vivo.
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http://dx.doi.org/10.1016/j.chembiol.2018.12.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6436940PMC
March 2019

Mitochondria as a therapeutic target for common pathologies.

Nat Rev Drug Discov 2018 12 5;17(12):865-886. Epub 2018 Nov 5.

WestCHEM School of Chemistry, University of Glasgow, Glasgow, UK.

Although the development of mitochondrial therapies has largely focused on diseases caused by mutations in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins, it has been found that mitochondrial dysfunction also contributes to the pathology of many common disorders, including neurodegeneration, metabolic disease, heart failure, ischaemia-reperfusion injury and protozoal infections. Mitochondria therefore represent an important drug target for these highly prevalent diseases. Several strategies aimed at therapeutically restoring mitochondrial function are emerging, and a small number of agents have entered clinical trials. This Review discusses the opportunities and challenges faced for the further development of mitochondrial pharmacology for common pathologies.
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http://dx.doi.org/10.1038/nrd.2018.174DOI Listing
December 2018

Mitochondrial superoxide generation induces a parkinsonian phenotype in zebrafish and huntingtin aggregation in human cells.

Free Radic Biol Med 2019 01 31;130:318-327. Epub 2018 Oct 31.

REQUIMTE/LAQV, Department of Drug Sciences, Pharmacology Lab, Faculty of Pharmacy, University of Porto, 4050-313 Porto, Portugal; Consortium for Mitochondrial Research (CfMR), University College London, Gower Street, WC1E 6BT London, UK. Electronic address:

Superoxide generation by mitochondria respiratory complexes is a major source of reactive oxygen species (ROS) which are capable of initiating redox signaling and oxidative damage. Current understanding of the role of mitochondrial ROS in health and disease has been limited by the lack of experimental strategies to selectively induce mitochondrial superoxide production. The recently-developed mitochondria-targeted redox cycler MitoParaquat (MitoPQ) overcomes this limitation, and has proven effective in vitro and in Drosophila. Here we present an in vivo study of MitoPQ in the vertebrate zebrafish model in the context of Parkinson's disease (PD), and in a human cell model of Huntington's disease (HD). We show that MitoPQ is 100-fold more potent than non-targeted paraquat in both cells and in zebrafish in vivo. Treatment with MitoPQ induced a parkinsonian phenotype in zebrafish larvae, with decreased sensorimotor reflexes, spontaneous movement and brain tyrosine hydroxylase (TH) levels, without detectable effects on heart rate or atrioventricular coordination. Motor phenotypes and TH levels were partly rescued with antioxidant or monoaminergic potentiation strategies. In a HD cell model, MitoPQ promoted mutant huntingtin aggregation without increasing cell death, contrasting with the complex I inhibitor rotenone that increased death in cells expressing either wild-type or mutant huntingtin. These results show that MitoPQ is a valuable tool for cellular and in vivo studies of the role of mitochondrial superoxide generation in redox biology, and as a trigger or co-stressor to model metabolic and neurodegenerative disease phenotypes.
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http://dx.doi.org/10.1016/j.freeradbiomed.2018.10.446DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6340810PMC
January 2019

Decreased mitochondrial metabolic requirements in fasting animals carry an oxidative cost.

Funct Ecol 2018 Sep 29;32(9):2149-2157. Epub 2018 May 29.

Institute of Biodiversity, Animal Health and Comparative Medicine University of Glasgow Glasgow UK.

Many animals experience periods of food shortage in their natural environment. It has been hypothesised that the metabolic responses of animals to naturally-occurring periods of food deprivation may have long-term negative impacts on their subsequent life-history.In particular, reductions in energy requirements in response to fasting may help preserve limited resources but potentially come at a cost of increased oxidative stress. However, little is known about this trade-off since studies of energy metabolism are generally conducted separately from those of oxidative stress.Using a novel approach that combines measurements of mitochondrial function with in vivo levels of hydrogen peroxide (HO) in brown trout (), we show here that fasting induces energy savings in a highly metabolically active organ (the liver) but at the cost of a significant increase in HO, an important form of reactive oxygen species (ROS).After a 2-week period of fasting, brown trout reduced their whole-liver mitochondrial respiratory capacities (state 3, state 4 and cytochrome oxidase activity), mainly due to reductions in liver size (and hence the total mitochondrial content). This was compensated for at the level of the mitochondrion, with an increase in state 3 respiration combined with a decrease in state 4 respiration, suggesting a selective increase in the capacity to produce ATP without a concomitant increase in energy dissipated through proton leakage However, the reduction in total hepatic metabolic capacity in fasted fish was associated with an almost two-fold increase in in vivo mitochondrial HO levels (as measured by the MitoB probe).The resulting increase in mitochondrial ROS, and hence potential risk of oxidative damage, provides mechanistic insight into the trade-off between the short-term energetic benefits of reducing metabolism in response to fasting and the potential long-term costs to subsequent life-history traits.
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http://dx.doi.org/10.1111/1365-2435.13125DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175143PMC
September 2018

Assessing the accuracy of intracameral phenylephrine preparation in cataract surgery.

Eye (Lond) 2018 10 15;32(10):1615-1620. Epub 2018 Jun 15.

Tennent Institute of Ophthalmology, Gartnavel General Hospital, 1053 Great Western Road, Glasgow, G12 0YN, UK.

Purpose: Unpreserved phenylephrine is often used as an off-licence intracameral surgical adjunct during cataract surgery to assist with pupil dilation and/or stabilise the iris in floppy iris syndrome. It can be delivered as a neat 0.2 ml bolus of either 2.5 or 10% strength, or in a range of ad-hoc dilutions. We wished to assess the accuracy of intracameral phenylephrine preparation in clinical practice.

Methods: Phenylephrine 0.2 ml was analysed both neat (2.5 and 10%) and in diluted form (ratio of 1:1 and 1:3). Samples were analysed using the validated spectrophotometric method.

Results: A total of 36 samples were analysed. The standard curve showed linearity for phenylephrine (R = 0.99). Wide variability was observed across all dilution groups. There was evidence of significant differences in the percentage deviations from intended results between dilutions (p < 0.001). Mean percentage deviation for 1:3 dilution was significantly greater than neat (p = 0.003) and 1:1 dilution (p = 0.001). There was no evidence of a significant difference between 1:1 and neat (p = 0.827).

Conclusions: Current ad-hoc dilution methods used to prepare intracameral phenylephrine are inaccurate and highly variable. Small volume 1 ml syringes should not be used for mixing or dilution of drug. Commercial intracameral phenylephrine products would address dosage concerns and could improve surgical outcomes in cases of poor pupil dilation and/or floppy iris syndrome.
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http://dx.doi.org/10.1038/s41433-018-0143-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6189205PMC
October 2018

Vascular Nox (NADPH Oxidase) Compartmentalization, Protein Hyperoxidation, and Endoplasmic Reticulum Stress Response in Hypertension.

Hypertension 2018 07 29;72(1):235-246. Epub 2018 May 29.

From the Institute of Cardiovascular and Medical Sciences (L.L.C., A.P.H., F.J.R., S.T., D.G., A.C.M., R.M.T.)

Vascular Nox (NADPH oxidase)-derived reactive oxygen species and endoplasmic reticulum (ER) stress have been implicated in hypertension. However, relationships between these processes are unclear. We hypothesized that Nox isoforms localize in a subcellular compartment-specific manner, contributing to oxidative and ER stress, which influence the oxidative proteome and vascular function in hypertension. Nox compartmentalization (cell fractionation), O (lucigenin), HO (amplex red), reversible protein oxidation (sulfenylation), irreversible protein oxidation (protein tyrosine phosphatase, peroxiredoxin oxidation), and ER stress (PERK [protein kinase RNA-like endoplasmic reticulum kinase], IRE1α [inositol-requiring enzyme 1], and phosphorylation/oxidation) were studied in spontaneously hypertensive rat (SHR) vascular smooth muscle cells (VSMCs). VSMC proliferation was measured by fluorescence-activated cell sorting, and vascular reactivity assessed in stroke-prone SHR arteries by myography. Noxs were downregulated by short interfering RNA and pharmacologically. In SHR, Noxs were localized in specific subcellular regions: Nox1 in plasma membrane and Nox4 in ER. In SHR, oxidative stress was associated with increased protein sulfenylation and hyperoxidation of protein tyrosine phosphatases and peroxiredoxins. Inhibition of Nox1 (NoxA1ds), Nox1/4 (GKT137831), and ER stress (4-phenylbutyric acid/tauroursodeoxycholic acid) normalized SHR vascular reactive oxygen species generation. GKT137831 reduced IRE1α sulfenylation and XBP1 (X-box binding protein 1) splicing in SHR. Increased VSMC proliferation in SHR was normalized by GKT137831, 4-phenylbutyric acid, and STF083010 (IRE1-XBP1 disruptor). Hypercontractility in the stroke-prone SHR was attenuated by 4-phenylbutyric acid. We demonstrate that protein hyperoxidation in hypertension is associated with oxidative and ER stress through upregulation of plasmalemmal-Nox1 and ER-Nox4. The IRE1-XBP1 pathway of the ER stress response is regulated by Nox4/reactive oxygen species and plays a role in the hyperproliferative VSMC phenotype in SHR. Our study highlights the importance of Nox subcellular compartmentalization and interplay between cytoplasmic reactive oxygen species and ER stress response, which contribute to the VSMC oxidative proteome and vascular dysfunction in hypertension.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.118.10824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004120PMC
July 2018

Mitochondrial oxidative stress causes insulin resistance without disrupting oxidative phosphorylation.

J Biol Chem 2018 05 29;293(19):7315-7328. Epub 2018 Mar 29.

Charles Perkins Centre, School of Life and Environmental Sciences, Camperdown, New South Wales 2006, Australia; Charles Perkins Centre, Sydney Medical School, University of Sydney, Camperdown, New South Wales 2006, Australia. Electronic address:

Mitochondrial oxidative stress, mitochondrial dysfunction, or both have been implicated in insulin resistance. However, disentangling the individual roles of these processes in insulin resistance has been difficult because they often occur in tandem, and tools that selectively increase oxidant production without impairing mitochondrial respiration have been lacking. Using the dimer/monomer status of peroxiredoxin isoforms as an indicator of compartmental hydrogen peroxide burden, we provide evidence that oxidative stress is localized to mitochondria in insulin-resistant 3T3-L1 adipocytes and adipose tissue from mice. To dissociate oxidative stress from impaired oxidative phosphorylation and study whether mitochondrial oxidative stress can cause insulin resistance, we used mitochondria-targeted paraquat (MitoPQ) to generate superoxide within mitochondria without directly disrupting the respiratory chain. At ≤10 μm, MitoPQ specifically increased mitochondrial superoxide and hydrogen peroxide without altering mitochondrial respiration in intact cells. Under these conditions, MitoPQ impaired insulin-stimulated glucose uptake and glucose transporter 4 (GLUT4) translocation to the plasma membrane in both adipocytes and myotubes. MitoPQ recapitulated many features of insulin resistance found in other experimental models, including increased oxidants in mitochondria but not cytosol; a more profound effect on glucose transport than on other insulin-regulated processes, such as protein synthesis and lipolysis; an absence of overt defects in insulin signaling; and defective insulin- but not AMP-activated protein kinase (AMPK)-regulated GLUT4 translocation. We conclude that elevated mitochondrial oxidants rapidly impair insulin-regulated GLUT4 translocation and significantly contribute to insulin resistance and that MitoPQ is an ideal tool for studying the link between mitochondrial oxidative stress and regulated GLUT4 trafficking.
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http://dx.doi.org/10.1074/jbc.RA117.001254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5950018PMC
May 2018

Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1.

Nature 2018 04 28;556(7699):113-117. Epub 2018 Mar 28.

Department of Cancer Biology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

The endogenous metabolite itaconate has recently emerged as a regulator of macrophage function, but its precise mechanism of action remains poorly understood. Here we show that itaconate is required for the activation of the anti-inflammatory transcription factor Nrf2 (also known as NFE2L2) by lipopolysaccharide in mouse and human macrophages. We find that itaconate directly modifies proteins via alkylation of cysteine residues. Itaconate alkylates cysteine residues 151, 257, 288, 273 and 297 on the protein KEAP1, enabling Nrf2 to increase the expression of downstream genes with anti-oxidant and anti-inflammatory capacities. The activation of Nrf2 is required for the anti-inflammatory action of itaconate. We describe the use of a new cell-permeable itaconate derivative, 4-octyl itaconate, which is protective against lipopolysaccharide-induced lethality in vivo and decreases cytokine production. We show that type I interferons boost the expression of Irg1 (also known as Acod1) and itaconate production. Furthermore, we find that itaconate production limits the type I interferon response, indicating a negative feedback loop that involves interferons and itaconate. Our findings demonstrate that itaconate is a crucial anti-inflammatory metabolite that acts via Nrf2 to limit inflammation and modulate type I interferons.
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http://dx.doi.org/10.1038/nature25986DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047741PMC
April 2018

Methionine sulfoxide reductase B3 requires resolving cysteine residues for full activity and can act as a stereospecific methionine oxidase.

Biochem J 2018 02 28;475(4):827-838. Epub 2018 Feb 28.

Institute of Molecular, Cellular and Systems Biology, CMVLS, University of Glasgow, Davidson Building, Glasgow G12 8QQ, U.K.

The oxidation of methionine residues in proteins occurs during oxidative stress and can lead to an alteration in protein function. The enzyme methionine sulfoxide reductase (Msr) reverses this modification. Here, we characterise the mammalian enzyme Msr B3. There are two splice variants of this enzyme that differ only in their N-terminal signal sequence, which directs the protein to either the endoplasmic reticulum (ER) or mitochondria. We demonstrate here that the enzyme can complement a bacterial strain, which is dependent on methionine sulfoxide reduction for growth, that the purified recombinant protein is enzymatically active showing stereospecificity towards -methionine sulfoxide, and identify the active site and two resolving cysteine residues. The enzyme is efficiently recycled by thioredoxin only in the presence of both resolving cysteine residues. These results show that for this isoform of Msrs, the reduction cycle most likely proceeds through a three-step process. This involves an initial sulfenylation of the active site thiol followed by the formation of an intrachain disulfide with a resolving thiol group and completed by the reduction of this disulfide by a thioredoxin-like protein to regenerate the active site thiol. Interestingly, the enzyme can also act as an oxidase catalysing the stereospecific formation of -methionine sulfoxide. This result has important implications for the role of this enzyme in the reversible modification of ER and mitochondrial proteins.
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http://dx.doi.org/10.1042/BCJ20170929DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6488974PMC
February 2018

MitoNeoD: A Mitochondria-Targeted Superoxide Probe.

Cell Chem Biol 2017 Oct 7;24(10):1285-1298.e12. Epub 2017 Sep 7.

WestCHEM School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK. Electronic address:

Mitochondrial superoxide (O) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O probe, MitoNeoD, which can assess O changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O production in health and disease.
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http://dx.doi.org/10.1016/j.chembiol.2017.08.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6278870PMC
October 2017

Characterisation of the biflavonoid hinokiflavone as a pre-mRNA splicing modulator that inhibits SENP.

Elife 2017 09 8;6. Epub 2017 Sep 8.

Centre for Gene Regulation and Expression, School of Life Sciences, University of Dundee, Dundee, United Kingdom.

We have identified the plant biflavonoid hinokiflavone as an inhibitor of splicing in vitro and modulator of alternative splicing in cells. Chemical synthesis confirms hinokiflavone is the active molecule. Hinokiflavone inhibits splicing in vitro by blocking spliceosome assembly, preventing formation of the B complex. Cells treated with hinokiflavone show altered subnuclear organization specifically of splicing factors required for A complex formation, which relocalize together with SUMO1 and SUMO2 into enlarged nuclear speckles containing polyadenylated RNA. Hinokiflavone increases protein SUMOylation levels, both in in vitro splicing reactions and in cells. Hinokiflavone also inhibited a purified, expressed SUMO protease, SENP1, in vitro, indicating the increase in SUMOylated proteins results primarily from inhibition of de-SUMOylation. Using a quantitative proteomics assay we identified many SUMO2 sites whose levels increased in cells following hinokiflavone treatment, with the major targets including six proteins that are components of the U2 snRNP and required for A complex formation.
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http://dx.doi.org/10.7554/eLife.27402DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619949PMC
September 2017

Assessment of HS using the newly developed mitochondria-targeted mass spectrometry probe MitoA.

J Biol Chem 2017 05 20;292(19):7761-7773. Epub 2017 Mar 20.

From the MRC Mitochondrial Biology Unit, University of Cambridge, Hills Road, Cambridge CB2 0XY, United Kingdom,

Hydrogen sulfide (HS) is produced endogenously and has multiple effects on signaling pathways and cell function. Mitochondria can be both an HS source and sink, and many of the biological effects of HS relate to its interactions with mitochondria. However, the significance of mitochondrial HS is uncertain, in part due to the difficulty of assessing changes in its concentration Although a number of fluorescent HS probes have been developed these are best suited to cells in culture and cannot be used To address this unmet need we have developed a mitochondria-targeted HS probe, MitoA, which can be used to assess relative changes in mitochondrial HS levels MitoA comprises a lipophilic triphenylphosphonium (TPP) cation coupled to an aryl azide. The TPP cation leads to the accumulation of MitoA inside mitochondria within tissues There, the aryl azido group reacts with HS to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial HS Both compounds can be detected sensitively by liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of the tissues, and quantified relative to deuterated internal standards. Here we describe the synthesis and characterization of MitoA and show that it can be used to assess changes in mitochondrial HS levels As a proof of principle we used MitoA to show that HS levels increase during myocardial ischemia.
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http://dx.doi.org/10.1074/jbc.M117.784678DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5427258PMC
May 2017