With the recognition of the central role of mitochondria in apoptosis, there is a need to develop specific tools to manipulate mitochondrial function within cells. Here we report on the development of a novel antioxidant that selectively blocks mitochondrial oxidative damage, enabling the roles of mitochondrial oxidative stress in different types of cell death to be inferred. This antioxidant, named mitoQ, is a ubiquinone derivative targeted to mitochondria by covalent attachment to a lipophilic triphenylphosphonium cation through an aliphatic carbon chain. Due to the large mitochondrial membrane potential, the cation was accumulated within mitochondria inside cells, where the ubiquinone moiety inserted into the lipid bilayer and was reduced by the respiratory chain. The ubiquinol derivative thus formed was an effective antioxidant that prevented lipid peroxidation and protected mitochondria from oxidative damage. After detoxifying a reactive oxygen species, the ubiquinol moiety was regenerated by the respiratory chain enabling its antioxidant activity to be recycled. In cell culture studies, the mitochondrially localized antioxidant protected mammalian cells from hydrogen peroxide-induced apoptosis but not from apoptosis induced by staurosporine or tumor necrosis factor-␣. This was compared with untargeted ubiquinone analogs, which were ineffective in preventing apoptosis. These results suggest that mitochondrial oxidative stress may be a critical step in apoptosis induced by hydrogen peroxide but not for apoptosis induced by staurosporine or tumor necrosis factor-␣. We have shown that selectively manipulating mitochondrial antioxidant status with targeted and recyclable antioxidants is a feasible approach to investigate the role of mitochondrial oxidative damage in apoptotic cell death. This approach will have further applications in investigating mitochondrial dysfunction in a range of experimental models.

Mitochondrial oxidative damage contributes to a range of degenerative diseases. Consequently, the selective inhibition of mitochondrial oxidative damage is a promising therapeutic strategy. One way to do this is to invent antioxidants that are selectively accumulated into mitochondria within patients. Such mitochondria-targeted antioxidants have been developed by conjugating the lipophilic triphenylphosphonium cation to an antioxidant moiety, such as ubiquinol or alpha-tocopherol. These compounds pass easily through all biological membranes, including the blood-brain barrier, and into muscle cells and thus reach those tissues most affected by mitochondrial oxidative damage. Furthermore, because of their positive charge they are accumulated several-hundredfold within mitochondria driven by the membrane potential, enhancing the protection of mitochondria from oxidative damage. These compounds protect mitochondria from damage following oral delivery and may therefore form the basis for mitochondria-protective therapies. Here we review the background and work to date on this class of mitochondria-targeted antioxidants.

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 unclear1–6. 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.

Mitochondrial dysfunction contributes to many human degenerative diseases but specific treatments are hampered by the difficulty of delivering bioactive molecules to mitochondria in vivo. To overcome this problem we developed a strategy to target bioactive molecules to mitochondria by attachment to the lipophilic triphenylphosphonium cation through an alkyl linker. These molecules rapidly permeate lipid bilayers and, because of the large mitochondrial membrane potential (negative inside), accumulate several hundredfold inside isolated mitochondria and within mitochondria in cultured cells. To determine whether this strategy could lead to the development of mitochondria-specific therapies, we investigated the administration and tissue distribution in mice of simple alkyltriphenylphosphonium cations and of mitochondria-targeted antioxidants comprising a triphenylphosphonium cation coupled to a coenzyme Q or vitamin E derivative. Significant doses of these compounds could be fed safely to mice over long periods, coming to steady-state distributions within the heart, brain, liver, and muscle. Therefore, mitochondria-targeted bioactive molecules can be administered orally, leading to their accumulation at potentially therapeutic concentrations in those tissues most affected by mitochondrial dysfunction. This finding opens the way to the testing of mitochondria-specific therapies in mouse models of human degenerative diseases.