Oxidative stress and mitochondrial dysfunction have major roles in the pathophysiology of cardiovascular disease. Mitochondria are the predominant source of energy production in the heart. Mitochondrial dysfunction decreases ATP production, while increasing reactive oxygen species (ROS) production. Early mitochondrial dysfunction leads to a vicious cycle, where excess ROS damages the mitochondria, leading to increased ROS production and impaired ATP production, decreasing the heart’s energetic capacity. Therefore, targeted mitochondrial antioxidants are a promising therapeutic target for cardiovascular disease and identification of novel endogenous mitochondrial antioxidants could lead to the discovery of new therapeutic targets. ABCB10 is a mitochondrial transporter with an unknown substrate. Our laboratory showed that hearts from mice with a heterozygous deletion of ABCB10 had decreased cardiac function and increased oxidative stress after ischemia reperfusion. Administration of an antioxidant ameliorated these effects, indicating that ABCB10 may have antioxidant effects. Therefore, we hypothesize that ABCB10 protects cardiac myocytes against oxidative injury through antioxidant effects in the cytosol and/or mitochondria. To test this hypothesis, we overexpressed ABCB10 in isolated adult rat ventricular myocytes (ARVMs) and exposed these cells to an oxidative challenge. ABCB10 overexpression protected cardiac myocytes against an oxidative insult, increasing cell viability and decreasing cytosolic ROS levels. ABCB10 overexpression also increased levels of HO-1, a cardioprotective and antioxidant-generating enzyme. We generated cardiac-specific ABCB10 knockout mice to explore the role of cardiac ABCB10 in vivo. We found that cardiac-specific deletion of ABCB10 in mice caused early death and mitochondrial dysfunction, evidenced by increased mitochondrial ROS production and decreased mitochondrial oxygen consumption. Additionally, microarray gene analysis revealed that hearts from ABCB10 knockout mice downregulated mitochondrial transcription and translation processes, indicating impaired mitochondrial proteostasis. These findings demonstrate that ABCB10 exerts an antioxidant effect, due, at least in part, to upregulation of the HO-1 antioxidant system. The ABCB10 mitochondrial transporter is involved in regulating mitochondrial oxidant levels and proteostasis and may be a novel therapeutic target in states associated with oxidative stress.

ABCB10, or ATP binding cassette sub-family B member 10, is a protein localized in the mitochondrial inner membrane. It belongs to the ABC transporter family whose members are proteins that facilitate substrate transport across various biological membranes. It has been found that ABCB10 is required for normal heme biosynthesis during erythroid differentiation and also plays a role in protection against the damage caused by reactive oxygen species (ROS) production. This protective effect exists both in the erythrocyte development and in the heart recovery after the ischemia-reperfusion injury. However, as an ABC transporter, its transported substrates are not known, neither is the mechanism by which ABCB10 plays a role in protection against ROS damage. In this dissertation an 8-azido-ATP photolabeling system is established to study the ATP binding and hydrolysis properties of ABCB10. Using this approach, it is found that the conserved amino acid residues Gly497 and Lys498 in the Walker A motif of the nucleotide binding domain of ABCB10 are required for ATP binding. On the other hand, Gly602 in the C-loop motif and Glu624 in the end of the Walker B motif are necessary for ATP hydrolysis. In addition, most ABC transporters increase ATP hydrolysis in the presence of their substrates. Therefore, the 8-azido-ATP photolabeling system can be utilized to test potential substrates of ABCB10. Substances related to the heme biosynthesis such as δ-aminolevulinic acid (dALA) and the mitochondrial redox state such as oxidized glutathione (GSSG) and reduced glutathione (GSH) are tested for this purpose. The 8-azido-ATP photolabeling system shows that GSSG stimulates ATP hydrolysis without affecting ATP binding, whereas GSH decreases ATP binding. Further study shows that the nucleotide binding domain of ABCB10 is glutathionylated at the cysteine residue on the position 547 (Cys547), suggesting that GSH may modulate ABCB10 activity via the glutathionylation-regulated ATP binding. This is a first insight into the molecular mechanism by which the mitochondrial redox state, through the regulation by GSH and GSSG, can modulate ABCB10 activity. / 2016-09-01T00:00:00Z