Incorporating a Polyethyleneglycol Linker to Enhance the Hydrophilicity of Mitochondria-Targeted Triphenylphosphonium Constructs.

The targeting of bioactive molecules and probes to mitochondria can be achieved by coupling to the lipophilic triphenyl phosphonium (TPP) cation, which accumulates several hundred-fold within mitochondria in response to the mitochondrial membrane potential (Δψm ). Typically, a simple alkane links the TPP to its "cargo", increasing overall hydrophobicity. As it would be beneficial to enhance the water solubility of mitochondria-targeted compounds we explored the effects of replacing the alkyl linker with a polyethylene glycol (PEG). We found that the use of PEG led to compounds that were readily taken up by isolated mitochondria and by mitochondria inside cells. Within mitochondria the PEG linker greatly decreased adsorption of the TPP constructs to the matrix-facing face of the mitochondrial inner membrane. These findings will allow the distribution of mitochondria-targeted TPP compounds within mitochondria to be fine-tuned.

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

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.

Control of mitochondrial superoxide production by reverse electron transport at complex I.

The generation of mitochondrial superoxide (O2̇̄) by reverse electron transport (RET) at complex I causes oxidative damage in pathologies such as ischemia reperfusion injury, but also provides the precursor to H2O2 production in physiological mitochondrial redox signaling. Here, we quantified the factors that determine mitochondrial O2̇̄ production by RET in isolated heart mitochondria. Measuring mitochondrial H2O2 production at a range of proton-motive force (Δp) values and for several coenzyme Q (CoQ) and NADH pool redox states obtained with the uncoupler p-trifluoromethoxyphenylhydrazone, we show that O2̇̄ production by RET responds to changes in O2 concentration, the magnitude of Δp, and the redox states of the CoQ and NADH pools. Moreover, we determined how expressing the alternative oxidase from the tunicate Ciona intestinalis to oxidize the CoQ pool affected RET-mediated O2̇̄ production at complex I, underscoring the importance of the CoQ pool for mitochondrial O2̇̄ production by RET. An analysis of O2̇̄ production at complex I as a function of the thermodynamic forces driving RET at complex I revealed that many molecules that affect mitochondrial reactive oxygen species production do so by altering the overall thermodynamic driving forces of RET, rather than by directly acting on complex I. These findings clarify the factors controlling RET-mediated mitochondrial O2̇̄ production in both pathological and physiological conditions. We conclude that O2̇̄ production by RET is highly responsive to small changes in Δp and the CoQ redox state, indicating that complex I RET represents a major mode of mitochondrial redox signaling.

Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.

Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure.

Assessment of H2S in vivo using the newly developed mitochondria-targeted mass spectrometry probe MitoA.

Hydrogen sulfide (H2S) is produced endogenously in vivo and has multiple effects on signaling pathways and cell function. Mitochondria can be both an H2S source and sink, and many of the biological effects of H2S relate to its interactions with mitochondria. However, the significance of mitochondrial H2S is uncertain, in part due to the difficulty of assessing changes in its concentration in vivo Although a number of fluorescent H2S probes have been developed these are best suited to cells in culture and cannot be used in vivo To address this unmet need we have developed a mitochondria-targeted H2S probe, MitoA, which can be used to assess relative changes in mitochondrial H2S levels in vivo 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 in vivo There, the aryl azido group reacts with H2S to form an aryl amine (MitoN). The extent of conversion of MitoA to MitoN thus gives an indication of the levels of mitochondrial H2S in vivo 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 H2S levels in vivo As a proof of principle we used MitoA to show that H2S levels increase in vivo during myocardial ischemia.

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.

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50µM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50nM, 30min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.

A model of mitochondrial superoxide production during ischaemia-reperfusion injury for therapeutic development and mechanistic understanding.

Ischaemia-reperfusion (IR) injury is the paradoxical consequence of the rapid restoration of blood flow to an ischaemic organ. Although reperfusion is essential for tissue survival in conditions such as myocardial infarction and stroke, the excessive production of mitochondrial reactive oxygen species (ROS) upon reperfusion initiates the oxidative damage that underlies IR injury, by causing cell death and inflammation. This ROS production is caused by an accumulation of the mitochondrial metabolite succinate during ischaemia, followed by its rapid oxidation upon reperfusion by succinate dehydrogenase (SDH), driving superoxide production at complex I by reverse electron transport. Inhibitors of SDH, such as malonate, show therapeutic potential by decreasing succinate oxidation and superoxide production upon reperfusion. To better understand the mechanism of mitochondrial ROS production upon reperfusion and to assess potential therapies, we set up an in vitro model of IR injury. For this, isolated mitochondria were incubated anoxically with succinate to mimic ischaemia and then rapidly reoxygenated to replicate reperfusion, driving a burst of ROS formation. Using this system, we assess the factors that contribute to the magnitude of mitochondrial ROS production in heart, brain, and kidney mitochondria, as well as screening for inhibitors of succinate oxidation with therapeutic potential.

Inactivation of pyruvate dehydrogenase kinase 2 by mitochondrial reactive oxygen species.

Reactive oxygen species are byproducts of mitochondrial respiration and thus potential regulators of mitochondrial function. Pyruvate dehydrogenase kinase 2 (PDHK2) inhibits the pyruvate dehydrogenase complex, thereby regulating entry of carbohydrates into the tricarboxylic acid (TCA) cycle. Here we show that PDHK2 activity is inhibited by low levels of hydrogen peroxide (H(2)O(2)) generated by the respiratory chain. This occurs via reversible oxidation of cysteine residues 45 and 392 on PDHK2 and results in increased pyruvate dehydrogenase complex activity. H(2)O(2) derives from superoxide (O(2)(.)), and we show that conditions that inhibit PDHK2 also inactivate the TCA cycle enzyme, aconitase. These findings suggest that under conditions of high mitochondrial O(2)(.) production, such as may occur under nutrient excess and low ATP demand, the increase in O(2)() and H(2)O(2) may provide feedback signals to modulate mitochondrial metabolism.

Selective uncoupling of individual mitochondria within a cell using a mitochondria-targeted photoactivated protonophore.

Depolarization of an individual mitochondrion or small clusters of mitochondria within cells has been achieved using a photoactivatable probe. The probe is targeted to the matrix of the mitochondrion by an alkyltriphenylphosphonium lipophilic cation and releases the protonophore 2,4-dinitrophenol locally in predetermined regions in response to directed irradiation with UV light via a local photolysis system. This also provides a proof of principle for the general temporally and spatially controlled release of bioactive molecules, pharmacophores, or toxins to mitochondria with tissue, cell, or mitochondrion specificity.

A mitochondria-targeted S-nitrosothiol modulates respiration, nitrosates thiols, and protects against ischemia-reperfusion injury.

Nitric oxide (NO(*)) competitively inhibits oxygen consumption by mitochondria at cytochrome c oxidase and S-nitrosates thiol proteins. We developed mitochondria-targeted S-nitrosothiols (MitoSNOs) that selectively modulate and protect mitochondrial function. The exemplar MitoSNO1, produced by covalently linking an S-nitrosothiol to the lipophilic triphenylphosphonium cation, was rapidly and extensively accumulated within mitochondria, driven by the membrane potential, where it generated NO(*) and S-nitrosated thiol proteins. MitoSNO1-induced NO(*) production reversibly inhibited respiration at cytochrome c oxidase and increased extracellular oxygen concentration under hypoxic conditions. MitoSNO1 also caused vasorelaxation due to its NO(*) generation. Infusion of MitoSNO1 during reperfusion was protective against heart ischemia-reperfusion injury, consistent with a functional modification of mitochondrial proteins, such as complex I, following S-nitrosation. These results support the idea that selectively targeting NO(*) donors to mitochondria is an effective strategy to reversibly modulate respiration and to protect mitochondria against ischemia-reperfusion injury.

Rapid and selective generation of H2S within mitochondria protects against cardiac ischemia-reperfusion injury.

Mitochondria-targeted H2S donors are thought to protect against acute ischemia-reperfusion (IR) injury by releasing H2S that decreases oxidative damage. However, the rate of H2S release by current donors is too slow to be effective upon administration following reperfusion. To overcome this limitation here we develop a mitochondria-targeted agent, MitoPerSulf that very rapidly releases H2S within mitochondria. MitoPerSulf is quickly taken up by mitochondria, where it reacts with endogenous thiols to generate a persulfide intermediate that releases H2S. MitoPerSulf is acutely protective against cardiac IR injury in mice, due to the acute generation of H2S that inhibits respiration at cytochrome c oxidase thereby preventing mitochondrial superoxide production by lowering the membrane potential. Mitochondria-targeted agents that rapidly generate H2S are a new class of therapy for the acute treatment of IR injury.

Tetra-arylborate lipophilic anions as targeting groups.

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.

Quantification and identification of mitochondrial proteins containing vicinal dithiols.

Vicinal dithiols may play a role in mitochondrial antioxidant defences and in redox signalling. We quantified protein vicinal dithiols within mammalian mitochondria using the vicinal dithiol-specific reagent phenylarsine oxide (PAO). We found 5-15% of thiols exposed on mitochondrial proteins were vicinal dithiols and that these thiols were particularly sensitive to oxidation by hydrogen peroxide. To visualise these proteins we used PAO to block vicinal dithiols, followed by alkylation of other thiols with N-ethylmaleimide (NEM). The PAO was then removed with 2,3-dimercapto-1-propanesulfonic acid (DMPS) and the exposed vicinal dithiols were labelled with iodoacetamide-biotin. To identify these proteins, we developed a selective proteomic methodology, based on Redox difference in gel electrophoresis (Redox-DIGE). Vicinal dithiol proteins were selectively labelled with a red fluorescent thiol-reactive Cy5 maleimide and mixed with Cy3 maleimide labelled protein in which vicinal dithiols remained untagged. Individual proteins were resolved by 2D gel electrophoresis and fluorescent scanning revealed vicinal dithiol proteins by the increase in Cy5 red fluorescence. These proteins were identified by peptide mass fingerprinting and mass spectrometry. These findings are consistent with roles for mitochondrial vicinal dithiol proteins in antioxidant defence and redox signalling and these methodologies will enable these roles to be explored.

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

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.

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

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 pKa. 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.

Rapid and extensive uptake and activation of hydrophobic triphenylphosphonium cations within cells.

Mitochondria-targeted molecules comprising the lipophilic TPP (triphenylphosphonium) cation covalently linked to a hydrophobic bioactive moiety are used to modify and probe mitochondria in cells and in vivo. However, it is unclear how hydrophobicity affects the rate and extent of their uptake into mitochondria within cells, making it difficult to interpret experiments because their intracellular concentration in different compartments is uncertain. To address this issue, we compared the uptake into both isolated mitochondria and mitochondria within cells of two hydrophobic TPP derivatives, [3H]MitoQ (mitoquinone) and [3H]DecylTPP, with the more hydrophilic TPP cation [3H]TPMP (methyltriphenylphosphonium). Uptake of MitoQ by mitochondria and cells was described by the Nernst equation and was approximately 5-fold greater than that for TPMP, as a result of its greater binding within the mitochondrial matrix. DecylTPP was also taken up extensively by cells, indicating that increased hydrophobicity enhanced uptake. Both MitoQ and DecylTPP were taken up very rapidly into cells, reaching a steady state within 15 min, compared with approximately 8 h for TPMP. This far faster uptake was the result of the increased rate of passage of hydrophobic TPP molecules through the plasma membrane. Within cells MitoQ was predominantly located within mitochondria, where it was rapidly reduced to the ubiquinol form, consistent with its protective effects in cells and in vivo being due to the ubiquinol antioxidant. The strong influence of hydrophobicity on TPP cation uptake into mitochondria within cells facilitates the rational design of mitochondria-targeted compounds to report on and modify mitochondrial function in vivo.

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

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.