BCNU-induced gR2 DEFECT mediates S-glutathionylation of Complex I and respiratory uncoupling in myocardium

Abstract

A deficiency of mitochondrial glutathione reductase (or GR2) is capable of adversely affecting the reduction of GSSG and increasing mitochondrial oxidative stress. BCNU [1, 3-bis (2-chloroethyl)-1-nitrosourea] is an anticancer agent and known inhibitor of cytosolic GR ex vivo and in vivo. Here we tested the hypothesis that a BCNU-induced GR2 defect contributes to mitochondrial dysfunction and subsequent impairment of heart function. Intraperitoneal administration of BCNU (40 mg/kg) specifically inhibited GR2 activity by 79.8±2.7% in the mitochondria of rat heart. However, BCNU treatment modestly enhanced the activities of mitochondrial Complex I and other ETC components. The cardiac function of BCNU-treated rats was analyzed by echocardiography, revealing a systolic dysfunction associated with decreased ejection fraction, decreased cardiac output, and an increase in left ventricular internal dimension and left ventricular volume in systole. The respiratory control index of isolated mitochondria from the myocardium was moderately decreased after BCNU treatment, whereas NADH-linked uncoupling of oxygen consumption was significantly enhanced. Extracellular flux analysis to measure the fatty acid oxidation of myocytes indicated a 20% enhancement after BCNU treatment. When the mitochondria were immunoblotted with antibodies against GSH and UCP3, both protein S-glutathionylation of Complex I and expression of UCP3 were significantly up-regulated. Overexpression of SOD2 in the myocardium significantly reversed BCNU-induced GR2 inhibition and mitochondrial impairment. In conclusion, BCNU-mediated cardiotoxicity is characterized by the GR2 deficiency that negatively regulates heart function by impairing mitochondrial integrity, increasing oxidative stress with Complex I S-glutathionylation, and enhancing uncoupling of mitochondrial respiration.

Increased mitochondrial prooxidant activity mediates up-regulation of Complex I S-glutathionylation via protein thiyl radical in the murine heart of eNOS−/−

“…However, achieving this thermodynamic equilibrium would require a marked decline in the intracellular GSH/GSSG ratio ( i.e., to a 1/1 ratio to drive 50% conversion of protein to glutathionylated form) [51, 52], which can occur only under extreme conditions. Pharmacologic inhibition of glutathione reductase 2 intensively increases mitochondrial GSSG/GSH ratio, enhancing S-glutathionylation of Complex I in vivo via thermodynamic mechanism [53]. In the myocardium of eNOS −/− mice, enhanced Complex I S-glutathionylation occurs at a relative high GSH/GSSG ratio (Table I); therefore, the mechanism of thiol-disulfide exchange is not likely.…”

Measurement of Reactive Oxygen Species, Reactive Nitrogen Species, and Redox-Dependent Signaling in the Cardiovascular System

“…11–14 DMPO has also been used to assay for mitochondria-derived ${\mathrm{normalO}}_2^{·-}$ generation by EPR. 13,15 Because of the short half-life of DMPO/ • OOH (t 1/2 ≈45 seconds) and the possibility that the ${\mathrm{normalO}}_2^{·-}$ adduct can decompose to give an • OH adduct, a superoxide dismutase (SOD)–sensitive 4-line spectrum of DMPO/ • OH is often detected. Therefore, a suitable SOD, polyethylene glycol–conjugated SOD (PEG-SOD), or SOD mimetic must be used to confirm that the detected DMPO/ • OH is dependent on the formation of ${\mathrm{normalO}}_2^{·-}$.…”

“…14,24 Ex vivo EPR spin trapping with DMPO has been used recently to quantify mitochondria-derived ${\mathrm{normalO}}_2^{·-}$ in the rat heart. 15 DEPMPO has been used extensively to investigate molecular mechanisms of ROS generation mediated by enzymes such as endothelial NOS (eNOS), 25 NADPH oxidases, xanthine oxidase, 26 and the mitochondrial electron transport chain in vitro. 27–30 …”

Impairment of pH gradient and membrane potential mediates redox dysfunction in the mitochondria of the post-ischemic heart

“…Earlier studies with isolated mitochondria of rat and murine hearts indicated that mitochondria produce large amount of • O 2 − under oligomycin-induced state 4 conditions, but • O 2 − generation is diminished under conditions of ADP-mediated state 3 and FCCP-mediated uncoupling [20, 22]. The results thus lend support to the hypothesis that the proton motive force is high, and substantially contributes to • O 2 − production when the activity of F 1 F 0 ATP synthase (F 1 F 0 ATPase) is low (state 2 conditions) or F 1 F 0 ATPase is inhibited (state 4 conditions).…”