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Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

ABSTRACT: 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.

SUBMITTER: Chen CL

PROVIDER: S-EPMC8629835 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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:The mitochondrial electrochemical gradient (?p), which comprises the pH gradient (?pH) and the membrane potential (??), is crucial in controlling energy transduction. During myocardial ischemia and reperfusion (IR), mitochondrial dysfunction mediates superoxide (·O2-) and H2O2 overproduction leading to oxidative injury. However, the role of ?pH and ?? in post-ischemic injury is not fully established. Here we studied mitochondria from the risk region of rat hearts subjected to 30 min of coronary ligation and 24 h of reperfusion in vivo. In the presence of glutamate, malate and ADP, normal mitochondria (mitochondria of non-ischemic region, NR) exhibited a heightened state 3 oxygen consumption rate (OCR) and reduced ·O2- and H2O2 production when compared to state 2 conditions. Oligomycin (increases ?pH by inhibiting ATP synthase) increased ·O2- and H2O2 production in normal mitochondria, but not significantly in the mitochondria of the risk region (IR mitochondria or post-ischemic mitochondria), indicating that normal mitochondrial ·O2- and H2O2 generation is dependent on ?pH and that IR impaired the ?pH of normal mitochondria. Conversely, nigericin (dissipates ?pH) dramatically reduced ·O2- and H2O2 generation by normal mitochondria under state 4 conditions, and this nigericin quenching effect was less pronounced in IR mitochondria. Nigericin also increased mitochondrial OCR, and predisposed normal mitochondria to a more oxidized redox status assessed by increased oxidation of cyclic hydroxylamine, CM-H. IR mitochondria, although more oxidized than normal mitochondria, were not responsive to nigericin-induced CM-H oxidation, which is consistent with the result that IR induced ?pH impairment in normal mitochondria. Valinomycin, a K+ ionophore used to dissipate ??, drastically diminished ·O2- and H2O2 generation by normal mitochondria, but less pronounced effect on IR mitochondria under state 4 conditions, indicating that ?? also contributed to ·O2- generation by normal mitochondria and that IR mediated ?? impairment. However, there was no significant difference in valinomycin-induced CM-H oxidation between normal and IR mitochondria. In conclusion, under normal conditions the proton backpressure imposed by ?pH restricts electron flow, controls a limited amount of ·O2- generation, and results in a more reduced myocardium; however, IR causes ?pH impairment and prompts a more oxidized myocardium.

Project description:BackgroundHeme is an essential iron-containing molecule for cardiovascular physiology, but in excess it may increase oxidative stress. Failing human hearts have increased heme levels, with upregulation of the rate-limiting enzyme in heme synthesis, δ-aminolevulinic acid synthase 2 (ALAS2), which is normally not expressed in cardiomyocytes. We hypothesized that increased heme accumulation (through cardiac overexpression of ALAS2) leads to increased oxidative stress and cell death in the heart.Methods and resultsWe first showed that ALAS2 and heme levels are increased in the hearts of mice subjected to coronary ligation. To determine the causative role of increased heme in the development of heart failure, we generated transgenic mice with cardiac-specific overexpression of ALAS2. While ALAS2 transgenic mice have normal cardiac function at baseline, their hearts display increased heme content, higher oxidative stress, exacerbated cell death, and worsened cardiac function after coronary ligation compared to nontransgenic littermates. We confirmed in cultured cardiomyoblasts that the increased oxidative stress and cell death observed with ALAS2 overexpression is mediated by increased heme accumulation. Furthermore, knockdown of ALAS2 in cultured cardiomyoblasts exposed to hypoxia reversed the increases in heme content and cell death. Administration of the mitochondrial antioxidant MitoTempo to ALAS2-overexpressing cardiomyoblasts normalized the elevated oxidative stress and cell death levels to baseline, indicating that the effects of increased ALAS2 and heme are through elevated mitochondrial oxidative stress. The clinical relevance of these findings was supported by the finding of increased ALAS2 induction and heme accumulation in failing human hearts from patients with ischemic cardiomyopathy compared to nonischemic cardiomyopathy.ConclusionsHeme accumulation is detrimental to cardiac function under ischemic conditions, and reducing heme in the heart may be a novel approach for protection against the development of heart failure.

Project description:ObjectivesCerebral blood flow (CBF) measurements after endovascular therapy (EVT) for acute ischemic stroke are important to distinguish early secondary injury related to persisting ischemia from that related to reperfusion when considering clinical response and infarct growth.MethodsWe compare reperfusion quantified by the modified Thrombolysis in Cerebral Infarction Score (mTICI) with perfusion measured by MRI dynamic contrast-enhanced perfusion within 5 h of EVT anterior circulation stroke. MR perfusion (rCBF, rCBV, rTmax, rT0) and mTICI scores were included in a predictive model for change in NIHSS at 24 h and diffusion-weighted imaging (DWI) lesion growth (acute to 24 h MRI) using a machine learning RRELIEFF feature selection coupled with a support vector regression.ResultsFor all perfusion parameters, mean values within the acute infarct for the TICI-2b group (considered clinically good reperfusion) were not significantly different from those in the mTICI <2b (clinically poor reperfusion). However, there was a statistically significant difference in perfusion values within the acute infarct region of interest between the mTICI-3 group versus both mTICI-2b and <2b (p = 0.02). The features that made up the best predictive model for change in NIHSS and absolute DWI lesion volume change was rT0 within acute infarct ROI and admission CTA collaterals respectively. No other variables, including mTICI scores, were selected for these best models. The correlation coefficients (Root mean squared error) for the cross-validation were 0.47 (13.7) and 0.51 (5.7) for change in NIHSS and absolute DWI lesion volume change.ConclusionMR perfusion following EVT provides accurate physiological approach to understanding the relationship of CBF, clinical outcome, and DWI growth.Advances in knowledgeMR perfusion CBF acquired is a robust, objective reperfusion measurement providing following recanalization of the target occlusion which is critical to distinguish potential therapeutic harm from the failed technical success of EVT as well as improve the responsiveness of clinical trial outcomes to disease modification.

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Reperfusion mediates heme impairment with increased protein cysteine sulfonation of mitochondrial complex III in the post-ischemic heart.

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