Patrick T. Kang

Protein thiyl radical mediates S-glutathionylation of complex I.

Complex I is a critical site of O(2)(•-) production and the major host of reactive protein thiols in mitochondria. In response to oxidative stress, complex I protein thiols at the 51- and 75-kDa subunits are reversibly S-glutathionylated. The mechanism of complex I S-glutathionylation is mainly obtained from insight into GSSG-mediated thiol-disulfide exchange, which would require a dramatic decline in the GSH/GSSG ratio. Intrinsic complex I S-glutathionylation can be detected in the rat heart at a relatively high GSH/GSSG ratio (J. Chen et al., J. Biol. Chem. 285:3168-3180, 2010). Thus, we hypothesized that reactive thiyl radical is more likely to mediate protein S-glutathionylation of complex I. Here we employed immuno-spin trapping and tandem mass spectrometry (LC/MS/MS) to test the hypothesis in the 75-kDa subunit from S-glutathionylated complex I. Under the conditions of O(2)(•-) production in the presence of GSH, we detected complex I S-glutathionylation at Cys-226, Cys-367, and Cys-727 of the 75-kDa subunit. Addition of a radical trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), significantly decreased complex I S-glutathionylation and subsequently increased the protein radical adduct of complex I-DMPO as detected by immunoblotting using an anti-DMPO antibody. LC/MS/MS analysis indicated that Cys-226, Cys-554, and Cys-727 were involved in DMPO binding, confirming that formation of the complex I thiyl radical mediates S-glutathionylation. LC/MS/MS analysis also showed that Cys-554 and Cys-727 were S-sulfonated under conditions of O(2)(•-) generation in the absence of DMPO. In myocytes (HL-1 cell line) treated with menadione to trigger mitochondrial O(2)(•-) generation, complex I protein radical and S-glutathionylation were increased. Thus mediation of complex I S-glutathionylation by the protein thiyl radical provides a unique pathway for the redox regulation of mitochondrial function.

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

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.

Impaired coronary metabolic dilation in the metabolic syndrome is linked to mitochondrial dysfunction and mitochondrial DNA damage

Mitochondrial dysfunction in obesity and diabetes can be caused by excessive production of free radicals, which can damage mitochondrial DNA. Because mitochondrial DNA plays a key role in the production of ATP necessary for cardiac work, we hypothesized that mitochondrial dysfunction, induced by mitochondrial DNA damage, uncouples coronary blood flow from cardiac work. Myocardial blood flow (contrast echocardiography) was measured in Zucker lean (ZLN) and obese fatty (ZOF) rats during increased cardiac metabolism (product of heart rate and arterial pressure, i.v. norepinephrine). In ZLN increased metabolism augmented coronary blood flow, but in ZOF metabolic hyperemia was attenuated. Mitochondrial respiration was impaired and ROS production was greater in ZOF than ZLN. These were associated with mitochondrial DNA (mtDNA) damage in ZOF. To determine if coronary metabolic dilation, the hyperemic response induced by heightened cardiac metabolism, is linked to mitochondrial function we introduced recombinant proteins (intravenously or intraperitoneally) in ZLN and ZOF to fragment or repair mtDNA, respectively. Repair of mtDNA damage restored mitochondrial function and metabolic dilation, and reduced ROS production in ZOF; whereas induction of mtDNA damage in ZLN reduced mitochondrial function, increased ROS production, and attenuated metabolic dilation. Adequate metabolic dilation was also associated with the extracellular release of ADP, ATP, and H2O2 by cardiac myocytes; whereas myocytes from rats with impaired dilation released only H2O2. In conclusion, our results suggest that mitochondrial function plays a seminal role in connecting myocardial blood flow to metabolism, and integrity of mtDNA is central to this process.

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

In response to oxidative stress, mitochondrial Complex I is reversibly S-glutathionylated. We hypothesized that protein S-glutathionylation (PrSSG) of Complex I is mediated by a kinetic mechanism involving reactive protein thiyl radical (PrS(•)) and GSH in vivo. Previous studies have shown that in vitro S-glutathionylation of isolated Complex I at the 51 and 75-kDa subunits was detected under the conditions of (•)O2(-) production, and mass spectrometry confirmed that formation of Complex I PrS(•) mediates PrSSG. Exposure of myocytes to menadione resulted in enhanced Complex I PrSSG and PrS(•) (Kang et al., Free Radical Biol. Med.52:962-973; 2012). In this investigation, we tested our hypothesis in the murine heart of eNOS(-/-). The eNOS(-/-) mouse is known to be hypertensive and develops the pathological phenotype of progressive cardiac hypertrophy. The mitochondria isolated from the eNOS(-/-) myocardium exhibited a marked dysfunction with impaired state 3 respiration, a declining respiratory control index, and decreasing enzymatic activities of ETC components. Further biochemical analysis and EPR measurement indicated defective aconitase activity, a marked increase in (•)O2(-) generation activity, and a more oxidized physiological setting. These results suggest increasing prooxidant activity and subsequent oxidative stress in the mitochondria of the eNOS(-/-) murine heart. When Complex I from the mitochondria of the eNOS(-/-) murine heart was analyzed by immunospin trapping and probed with anti-GSH antibody, both PrS(•) and PrSSG of Complex I were significantly enhanced. Overexpression of SOD2 in the murine heart dramatically diminished the detected PrS(•), supporting the conclusion that mediation of Complex I PrSSG by oxidative stress-induced PrS(•) is a unique pathway for the redox regulation of mitochondrial function in vivo.

Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction

We demonstrated previously that TRPV1-dependent coupling of coronary blood flow (CBF) to metabolism is disrupted in diabetes. A critical amount of H2O2 contributes to CBF regulation; however, excessive H2O2 impairs responses. We sought to determine the extent to which differential regulation of TRPV1 by H2O2 modulates CBF and vascular reactivity in diabetes. We used contrast echocardiography to study TRPV1 knockout (V1KO), db/db diabetic, and wild type C57BKS/J (WT) mice. H2O2 dose-dependently increased CBF in WT mice, a response blocked by the TRPV1 antagonist SB366791. H2O2-induced vasodilation was significantly inhibited in db/db and V1KO mice. H2O2 caused robust SB366791-sensitive dilation in WT coronary microvessels; however, this response was attenuated in vessels from db/db and V1KO mice, suggesting H2O2-induced vasodilation occurs, in part, via TRPV1. Acute H2O2 exposure potentiated capsaicin-induced CBF responses and capsaicin-mediated vasodilation in WT mice, whereas prolonged luminal H2O2 exposure blunted capsaicin-induced vasodilation. Electrophysiology studies re-confirms acute H2O2 exposure activated TRPV1 in HEK293A and bovine aortic endothelial cells while establishing that H2O2 potentiate capsaicin-activated TRPV1 currents, whereas prolonged H2O2 exposure attenuated TRPV1 currents. Verification of H2O2-mediated activation of intrinsic TRPV1 specific currents were found in isolated mouse coronary endothelial cells from WT mice and decreased in endothelial cells from V1KO mice. These data suggest prolonged H2O2 exposure impairs TRPV1-dependent coronary vascular signaling. This may contribute to microvascular dysfunction and tissue perfusion deficits characteristic of diabetes.

Differential protein acetylation assists import of excess SOD2 into mitochondria and mediates SOD2 aggregation associated with cardiac hypertrophy in the murine SOD2-tg heart

Abstract SOD2 is the primary antioxidant enzyme neutralizing •O2− in mitochondria. Cardiac‐specific SOD2 overexpression (SOD2‐tg) induces supernormal function and cardiac hypertrophy in the mouse heart. However, the reductive stress imposed by SOD2 overexpression results in protein aggregation of SOD2 pentamers and differential hyperacetylation of SOD2 in the mitochondria and cytosol. Here, we studied SOD2 acetylation in SOD2‐tg and wild‐type mouse hearts. LC‐MS/MS analysis indicated the presence of four acetylated lysines in matrix SOD2 and nine acetylated lysines in cytosolic SOD2 from the SOD2‐tg heart. However, only one specific acetylated lysine residue was detected in the mitochondria of the wild‐type heart, which was consistent with Sirt3 downregulation in the SOD2‐tg heart. LC‐MS/MS further detected hyperacetylated SOD2 with a signaling peptide in the mitochondrial inner membrane and matrix of the SOD2‐tg heart, indicating partial arrest of the SOD2 precursor in the membrane during translocation into the mitochondria. Upregulation of HSP 70 and cytosolic HSP 60 enabled the translocation of excess SOD2 into mitochondria. In vitro acetylation of matrix SOD2 with Ac2O deaggregated pentameric SOD2, restored the profile of cytosolic SOD2 hyperacetylation, and decreased matrix SOD2 activity. As revealed by 3D structure, acetylation of K89, K134, and K154 of cytosolic SOD2 induces unfolding of the tertiary structure and breaking of the salt bridges that are important for the quaternary structure, suggesting that hyperacetylation and HSP 70 upregulation maintain the unfolded status of SOD2 in the cytosol and mediate the import of SOD2 across the membrane. Downregulation of Sirt3, HSP 60, and presequence protease in the mitochondria of the SOD2‐tg heart promoted protein misfolding that led to pentameric aggregation. Graphical abstract Figure. No Caption available. HighlightsCardiac‐specific overexpression of SOD2 induces hypertrophy in mouse heart.The myocardium of SOD2‐tg displays differential SOD2 acetylation and aggregate.Hyperacetylation of SOD2 mediates its mitochondrial translocation.Deacetylation of SOD2 mediates activation, misfolding, and protein aggregate.

Oxidative modifications of mitochondria complex II.

Increased superoxide (O2 (·-)) and nitric oxide (NO) production is a key mechanism of mitochondrial dysfunction in myocardial ischemia/reperfusion injury. In the complex II, oxidative impairment, decreased protein S-glutathionylation, and increased protein tyrosine nitration at the 70 kDa subunit occur in the post-ischemic myocardium (Zhang et al., Biochemistry 49:2529-2539, 2010; Chen et al., J Biol Chem 283:27991-28003, 2008; Chen et al., J Biol Chem 282: 32640-32654, 2007). To gain the deeper insights into ROS-mediated oxidative modifications relevant in myocardial infarction, isolated complex II is subjected to in vitro oxidative modifications with GSSG (to induce cysteine S-glutathionylation) or OONO(-) (to induce tyrosine nitration). Here, we describe the protocol to characterize the specific oxidative modifications at the 70 kDa subunit by nano-LC/MS/MS analysis. We further demonstrate the cellular oxidative modification with protein nitration/S-glutathionylation with immunofluorescence microscopy using the antibodies against 3-nitrotyrosine/glutathione and complex II 70 kDa polypeptide (AbGSC90) in myocytes under conditions of oxidative stress.

Mitochondrial complex I in the post-ischemic heart: reperfusion-mediated oxidative injury and protein cysteine sulfonation

A serious consequence of ischemia-reperfusion injury (I/R) is oxidative damage leading to mitochondrial dysfunction. Such I/R-induced mitochondrial dysfunction is observed as impaired state 3 respiration and overproduction of O2-. The cascading ROS can propagate cysteine oxidation on mitochondrial complex I and add insult to injury. Herein we employed LC-MS/MS to identify protein sulfonation of complex I in mitochondria from the infarct region of rat hearts subjected to 30-min of coronary ligation and 24-h of reperfusion in vivo as well as the mitochondria of sham controls. Mitochondrial preparations from the I/R regions had enhanced sulfonation levels on the cysteine ligands of iron‑sulfur clusters, including N3 (C425), N1b (C92), N4 (C226), N2 (C158/C188), and N1a (C134/C139). The 4Fe-4S centers of N3, N1b, N4, and N2 are key redox-active components of complex I, thus sulfonation of metal-binding sites impaired the main electron transfer pathway. The binuclear N1a has a very low redox potential and an antioxidative function. Increased C134/C139 sulfonation by I/R impaired the N1a cluster, potentially contributing to overall O2- generation by the FMN moiety of complex I. MS analysis also revealed I/R-mediated increased sulfonation at the core subunits of 51 kDa (C125, C187, C206, C238, C255, C286), 75 kDa (C367, C554, C564, C727), 49 kDa (C146, C326, C347), and PSST (C188). These results were consistent with the consensus indicating that 51 kDa and 75 kDa are two of major subunits hosting regulatory thiols, and their enhanced sulfonation by I/R predisposed the myocardium to further oxidant stress with impaired ubiquinone reduction. MS analysis further showed I/R-mediated enhanced sulfonation at the supernumerary subunits of 42 kDa (C67, C112, C183, C253), 15 kDa (C43), and 13 kDa (C79). The 42 kDa protein is metazoan-specific, which was reported to stabilize mammalian complex I. C43 of the 15 kDa subunit forms an intramolecular disulfide bond with C56, which was reported to stabilize complex I structure. C79 of the 13 kDa subunit is involved in Zn2+-binding, which was reported functionally important for complex I assembly. C79 sulfonation by I/R was found to impair Zn2+-binding. No significant enhancement of protein sulfonation was observed in mitochondrial complex I from the rat heart subjected to 30-min ischemia alone in vivo despite a decreased state 3 respiration, suggesting that the physiologic conditions of hyperoxygenation during reperfusion mediated an increase in complex I sulfonation and oxidative injury. In conclusion, sulfonation of specific cysteines of complex I mediates I/R-induced mitochondrial dysfunction via impaired ETC activity, increasing •O2- production, and mediating redox dysfunction of complex I.