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Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

ABSTRACT: It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity.SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of Met(80)-Fe ligation in ischemia. Mitochondria were isolated from ischemic rat kidneys to determine sites of respiratory inhibition. Mitochondrial cyt c and cyt c Met-O were quantified by western blot, and cristae architecture was examined by electron microscopy.Biochemical and structural studies showed that SS-20 selectively targets cardiolipin (CL) and protects Met(80)-Fe ligation in cyt c. Ischemic mitochondria showed 17-fold increase in Met-O cyt c, and dramatic cristaeolysis. Loss of cyt c was associated with proteolytic degradation of OPA1. Ischemia significantly inhibited ET initiated by direct reduction of cyt c and coupled respiration. All changes were prevented by SS-20.Our results show that ischemia disrupts the Met(80)-Fe ligation of cyt c resulting in the formation of a globin-like pentacoordinated heme Fe(2+) that inhibits ET, and converts cyt c into an oxygenase to cause CL peroxidation and proteolytic degradation of OPA1, resulting in cyt c release.Cyt c heme structure represents a novel target for minimizing ischemic injury. SS-20, which we show to selectively target CL and protect the Met(80)-Fe ligation, minimizes ischemic injury and promotes ATP recovery.

SUBMITTER: Birk AV

PROVIDER: S-EPMC4547887 | biostudies-literature | 2015 Oct

REPOSITORIES: biostudies-literature

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Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

Birk Alexander V AV Chao Wesley M WM Liu Shaoyi S Soong Yi Y Szeto Hazel H HH

Biochimica et biophysica acta 20150610 10

<h4>Background</h4>It was recently suggested that electron flow into cyt c, coupled with ROS generation, oxidizes cyt c Met(80) to Met(80) sulfoxide (Met-O) in isolated hearts after ischemia-reperfusion, and converts cyt c to a peroxidase. We hypothesize that ischemia disrupts Met(80)-Fe ligation of cyt c, forming pentacoordinated heme Fe(2+), which inhibits electron transport (ET) and promotes oxygenase activity.<h4>Methods</h4>SS-20 (Phe-D-Arg-Phe-Lys-NH2) was used to demonstrate the role of M ...[more]

PMID: 26071084

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Project description:BackgroundRenal ischemia-reperfusion injury (IRI) is a major factor contributing to acute kidney injury and it is associated with a high morbidity and mortality if untreated. Renal IRI depletes cellular and tissue adenosine triphosphate (ATP), which compromises mitochondrial function, further exacerbating renal tubular injury. Currently, no treatment for IRI is available. This study investigates the protective role of treprostinil in improving mitochondria biogenesis and recovery during rat renal IRI.MethodsMale Sprague Dawley rats were randomly assigned to groups: control, sham, IRI-placebo or IRI-treprostinil and subjected to 45 min of bilateral renal ischemia followed by 1-72 h reperfusion. Placebo or treprostinil (100 ng/kg/min) was administered subcutaneously via an osmotic minipump.ResultsTreprostinil significantly reduced peak elevated serum creatinine (SCr) levels and accelerated normalization relative to IRI-placebo (p < 0.0001). Treatment with treprostinil also inhibited IRI-mediated renal apoptosis, mitochondrial oxidative injury (p < 0.05), and the release of cytochrome c (p < 0.01) vs. IRI-placebo. In addition, treprostinil preserved renal mitochondrial DNA copy number (p < 0.0001) and renal ATP levels (p < 0.05) to nearly those of sham-operated animals. Non-targeted semi-quantitative proteomics showed reduced levels of ATP synthase subunits in the IRI-placebo group which were restored to sham levels by treprostinil treatment (p < 0.05). Furthermore, treprostinil reduced renal IRI-induced upregulated Drp1 and pErk protein levels, and restored Sirt3 and Pgc-1α levels to baseline (p < 0.05).ConclusionsTreprostinil reduces mitochondrial-mediated renal apoptosis, inhibits mitochondria fission, and promotes mitochondria fusion, thereby accelerating mitochondrial recovery and protecting renal proximal tubules from renal IRI. These results support the clinical investigation of treprostinil as a viable therapy to reduce renal IRI.

Project description:Reperfusion injury after extended ischemia accounts for approximately 50% of myocardial infarct size, and there is no standard therapy. HDAC inhibition reduces infarct size and enhances cardiomyocyte autophagy and PGC1α-mediated mitochondrial biogenesis when administered at the time of reperfusion. Furthermore, a specific autophagy-inducing peptide, Tat-Beclin 1 (TB), reduces infarct size when administered at the time of reperfusion. However, since SAHA affects multiple pathways in addition to inducing autophagy, whether autophagic flux induced by TB maintains mitochondrial homeostasis during ischemia/reperfusion (I/R) injury is unknown. We tested whether the augmentation of autophagic flux by TB has cardioprotection by preserving mitochondrial homeostasis both in vitro and in vivo. Wild-type mice were randomized into two groups: Tat-Scrambled (TS) peptide as the control and TB as the experimental group. Mice were subjected to I/R surgery (45 min coronary ligation, 24 h reperfusion). Autophagic flux, mitochondrial DNA (mtDNA), mitochondrial morphology, and mitochondrial dynamic genes were assayed. Cultured neonatal rat ventricular myocytes (NRVMs) were treated with a simulated I/R injury to verify cardiomyocyte specificity. The essential autophagy gene, ATG7, conditional cardiomyocyte-specific knockout (ATG7 cKO) mice, and isolated adult mouse ventricular myocytes (AMVMs) were used to evaluate the dependency of autophagy in adult cardiomyocytes. In NRVMs subjected to I/R, TB increased autophagic flux, mtDNA content, mitochondrial function, reduced reactive oxygen species (ROS), and mtDNA damage. Similarly, in the infarct border zone of the mouse heart, TB induced autophagy, increased mitochondrial size and mtDNA content, and promoted the expression of PGC1α and mitochondrial dynamic genes. Conversely, loss of ATG7 in AMVMs and in the myocardium of ATG7 cKO mice abolished the beneficial effects of TB on mitochondrial homeostasis. Thus, autophagic flux is a sufficient and essential process to mitigate myocardial reperfusion injury by maintaining mitochondrial homeostasis and partly by inducing PGC1α-mediated mitochondrial biogenesis.

Project description:AimsThe FDA-approved histone deacetylase (HDAC) inhibitor, suberoylanilide hydroxamic acid (SAHA, Vorinostat) has been shown to induce cardiomyocyte autophagy and blunt ischemia/reperfusion (I/R) injury when administered at the time of reperfusion. However, the precise mechanisms underlying the cardioprotective activity of SAHA are unknown. Mitochondrial dysfunction and oxidative damage are major contributors to myocardial apoptosis during I/R injury. We hypothesize that SAHA protects the myocardium by maintaining mitochondrial homeostasis and reducing reactive oxygen species (ROS) production during I/R injury.MethodsMouse and cultured cardiomyocytes (neonatal rat ventricular myocytes and human embryonic stem cell-derived cardiomyocytes) I/R models were used to investigate the effects of SAHA on mitochondria. ATG7 knockout mice, ATG7 knockdown by siRNA and PGC-1α knockdown by adenovirus in cardiomyocytes were used to test the dependency of autophagy and PGC-1α-mediated mitochondrial biogenesis respectively.ResultsIntact and total mitochondrial DNA (mtDNA) content and mitochondrial mass were significantly increased in cardiomyocytes by SAHA pretreatment before simulated I/R. In vivo, I/R induced >50% loss of mtDNA content in the border zones of mouse hearts, but SAHA pretreatment and reperfusion treatment alone reverted mtDNA content and mitochondrial mass to control levels. Moreover, pretreatment of cardiomyocytes with SAHA resulted in a 4-fold decrease in I/R-induced loss of mitochondrial membrane potential and a 25%-40% reduction in cytosolic ROS levels. However, loss-of-function of ATG7 in cardiomyocytes or mouse myocardium abolished the protective effects of SAHA on ROS levels, mitochondrial membrane potential, mtDNA levels, and mitochondrial mass. Lastly, PGC-1α gene expression was induced by SAHA in NRVMs and mouse heart subjected to I/R, and loss of PGC-1α abrogated SAHA's mitochondrial protective effects in cardiomyocytes.ConclusionsSAHA prevents I/R induced-mitochondrial dysfunction and loss, and reduces myocardial ROS production when given before or after the ischemia. The protective effects of SAHA on mitochondria are dependent on autophagy and PGC-1α-mediated mitochondrial biogenesis.

Project description:Background: Ischemia reperfusion injury (IRI) plays a major role in solid organ transplantation. The length of warm ischemia time is critical for the extent of tissue damage in renal IRI. In this experimental study we hypothesized that local release of labile heme in renal tissue is triggered by the duration of warm ischemia (15 vs. 45 min IRI) and mediates complement activation, cytokine release, and inflammation. Methods: To induce IRI, renal pedicle clamping was performed in male C57BL/6 mice for short (15 min) or prolonged (45 min) time periods. Two and 24 h after experimental ischemia tissue injury labile heme levels in the kidney were determined with an apo-horseradish peroxidase assay. Moreover, renal injury, cytokines, and C5a and C3a receptor (C5aR, C3aR) expression were determined by histology, immunohistochemistry and qPCR, respectively. In addition, in vitro studies stimulating bone marrow-derived macrophages with LPS and the combination of LPS and heme were performed and cytokine expression was measured. Results: Inflammation and local tissue injury correlated with the duration of warm ischemia time. Labile heme concentrations in renal tissue were significantly higher after prolonged (45 min) as compared to short (15 min) IRI. Notably, expression of the inducible heme-degrading enzyme heme oxygenase-1 (HO-1) was up-regulated in kidneys after prolonged, but not after short IRI. C5aR, the pro-inflammatory cytokines IL-6 and TNF-α as well as pERK were up-regulated after prolonged, but not after short ischemia times. Consecutively, neutrophil infiltration and up-regulation of pro-fibrotic cytokines such as CTGF and PAI were more pronounced in prolonged IRI in comparison to short IRI. In vitro stimulation of macrophages with LPS revealed that IL-6 expression was enhanced in the presence of heme. Finally, administration of the heme scavenger human serum albumin (HSA) reduced the expression of pro-inflammatory cytokines, C3a receptor and improved tubular function indicated by enhanced alpha 1 microglobulin (A1M) absorption after IRI. Conclusions: Our data show that prolonged duration of warm ischemia time increased labile heme levels in the kidney, which correlates with IRI-dependent inflammation and up-regulation of anaphylatoxin receptor expression.

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Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

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Disruption of cytochrome c heme coordination is responsible for mitochondrial injury during ischemia.

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