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DJ-1 attenuates the glycation of mitochondrial complex I and complex III in the post-ischemic heart.

ABSTRACT: DJ-1 is a ubiquitously expressed protein that protects cells from stress through its conversion into an active protease. Recent work found that the active form of DJ-1 was induced in the ischemic heart as an endogenous mechanism to attenuate glycative stress-the non-enzymatic glycosylation of proteins. However, specific proteins protected from glycative stress by DJ-1 are not known. Given that mitochondrial electron transport proteins have a propensity for being targets of glycative stress, we investigated if DJ-1 regulates the glycation of Complex I and Complex III after myocardial ischemia-reperfusion (I/R) injury. Initial studies found that DJ-1 localized to the mitochondria and increased its interaction with Complex I and Complex III 3 days after the onset of myocardial I/R injury. Next, we investigated the role DJ-1 plays in modulating glycative stress in the mitochondria. Analysis revealed that compared to wild-type control mice, mitochondria from DJ-1 deficient (DJ-1 KO) hearts showed increased levels of glycative stress following I/R. Additionally, Complex I and Complex III glycation were found to be at higher levels in DJ-1 KO hearts. This corresponded with reduced complex activities, as well as reduced mitochondrial oxygen consumption ant ATP synthesis in the presence of pyruvate and malate. To further determine if DJ-1 influenced the glycation of the complexes, an adenoviral approach was used to over-express the active form of DJ-1(AAV9-DJ1ΔC). Under I/R conditions, the glycation of Complex I and Complex III were attenuated in hearts treated with AAV9-DJ1ΔC. This was accompanied by improvements in complex activities, oxygen consumption, and ATP production. Together, this data suggests that cardiac DJ-1 maintains Complex I and Complex III efficiency and mitochondrial function during the recovery from I/R injury. In elucidating a specific mechanism for DJ-1's role in the post-ischemic heart, these data break new ground for potential therapeutic strategies using DJ-1 as a target.

SUBMITTER: Pantner Y

PROVIDER: S-EPMC8484662 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:A serious consequence of myocardial ischemia-reperfusion injury (I/R) is oxidative damage, which causes mitochondrial dysfunction. The cascading ROS can propagate and potentially induce heme bleaching and protein cysteine sulfonation (PrSO3H) of the mitochondrial electron transport chain. Herein we studied the mechanism of I/R-mediated irreversible oxidative injury of complex III in mitochondria from rat hearts subjected to 30-min of ischemia and 24-h of reperfusion in vivo. In the I/R region, the catalytic activity of complex III was significantly impaired. Spectroscopic analysis indicated that I/R mediated the destruction of hemes b and c + c1 in the mitochondria, supporting I/R-mediated complex III impairment. However, no significant impairment of complex III activity and heme damage were observed in mitochondria from the risk region of rat hearts subjected only to 30-min ischemia, despite a decreased state 3 respiration. In the I/R mitochondria, carbamidomethylated C122/C125 of cytochrome c1 via alkylating complex III with a down regulation of HCCS was exclusively detected, supporting I/R-mediated thioether defect of heme c1. LC-MS/MS analysis showed that I/R mitochondria had intensely increased complex III PrSO3H levels at the C236 ligand of the [2Fe2S] cluster of the Rieske iron‑sulfur protein (uqcrfs1), thus impairing the electron transport activity. MS analysis also indicated increased PrSO3H of the hinge protein at C65 and of cytochrome c1 at C140 and C220, which are confined in the intermembrane space. MS analysis also showed that I/R extensively enhanced the PrSO3H of the core 1 (uqcrc1) and core 2 (uqcrc2) subunits in the matrix compartment, thus supporting the conclusion that complex III releases ROS to both sides of the inner membrane during reperfusion. Analysis of ischemic mitochondria indicated a modest reduction from the basal level of complex III PrSO3H detected in the mitochondria of sham control hearts, suggesting that the physiologic hyperoxygenation and ROS overproduction during reperfusion mediated the enhancement of complex III PrSO3H. In conclusion, reperfusion-mediated heme damage with increased PrSO3H controls oxidative injury to complex III and aggravates mitochondrial dysfunction in the post-ischemic heart.

Project description: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.

Project description:BackgroundAbnormalities in cardiac energy metabolism occur in heart failure (HF) and contribute to contractile dysfunction, but their role, if any, in HF-related pathologic remodeling is much less established. CK (creatine kinase), the primary muscle energy reserve reaction which rapidly provides ATP at the myofibrils and regenerates mitochondrial ADP, is down-regulated in experimental and human HF. We tested the hypotheses that pathologic remodeling in human HF is related to impaired cardiac CK energy metabolism and that rescuing CK attenuates maladaptive hypertrophy in experimental HF.MethodsFirst, in 27 HF patients and 14 healthy subjects, we measured cardiac energetics and left ventricular remodeling using noninvasive magnetic resonance 31P spectroscopy and magnetic resonance imaging, respectively. Second, we tested the impact of metabolic rescue with cardiac-specific overexpression of either Ckmyofib (myofibrillar CK) or Ckmito (mitochondrial CK) on HF-related maladaptive hypertrophy in mice.ResultsIn people, pathologic left ventricular hypertrophy and dilatation correlate closely with reduced myocardial ATP levels and rates of ATP synthesis through CK. In mice, transverse aortic constriction-induced left ventricular hypertrophy and dilatation are attenuated by overexpression of CKmito, but not by overexpression of CKmyofib. CKmito overexpression also attenuates hypertrophy after chronic isoproterenol stimulation. CKmito lowers mitochondrial reactive oxygen species, tissue reactive oxygen species levels, and upregulates antioxidants and their promoters. When the CK capacity of CKmito-overexpressing mice is limited by creatine substrate depletion, the protection against pathologic remodeling is lost, suggesting the ADP regenerating capacity of the CKmito reaction rather than CK protein per se is critical in limiting adverse HF remodeling.ConclusionsIn the failing human heart, pathologic hypertrophy and adverse remodeling are closely related to deficits in ATP levels and in the CK energy reserve reaction. CKmito, sitting at the intersection of cardiac energetics and redox balance, plays a crucial role in attenuating pathologic remodeling in HF. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT00181259.

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DJ-1 attenuates the glycation of mitochondrial complex I and complex III in the post-ischemic heart.

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