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Succinate metabolism: a new therapeutic target for myocardial reperfusion injury.
Myocardial ischaemia/reperfusion (IR) injury is a major cause of death worldwide and remains a disease for which current clinical therapies are strikingly deficient. While the production of mitochondrial reactive oxygen species (ROS) is a critical driver of tissue damage upon reperfusion, the precise mechanisms underlying ROS production have remained elusive. More recently, it has been demonstrated that a specific metabolic mechanism occurs during ischaemia that underlies elevated ROS at reperfusion, suggesting a unifying model as to why so many different compounds have been found to be cardioprotective against IR injury. This review will discuss the role of the citric acid cycle intermediate succinate in IR pathology focusing on the mechanism by which this metabolite accumulates during ischaemia and how it can drive ROS production at Complex I via reverse electron transport. We will then examine the potential for manipulating succinate accumulation and metabolism during IR injury in order to protect the heart against IR damage and discuss targets for novel therapeutics designed to reduce reperfusion injury in patients
Moving Forwards by Blocking Back-Flow: The Yin and Yang of MI Therapy.
Mitochondrial reactive oxygen species production has emerged as an important pathological mechanism in myocardial ischemia/reperfusion injury. Attempts at targeting reactive oxygen species by scavenging using antioxidants have, however, been clinically disappointing. This review will provide an overview of the current understanding of mitochondrial reactive oxygen species in ischemia/reperfusion injury. We will outline novel therapeutic approaches designed to directly target the mitochondrial respiratory chain and prevent excessive reactive oxygen species production and its associated pathology. This approach could lead to more effective interventions in an area where there is an urgent need for new treatments.Work in our laboratories is supported by the Medical Research Council (UK) and the British Heart Foundation.This is the author accepted manuscript. The final version is available from the American Heart Association via http://dx.doi.org/10.1161/CIRCRESAHA.115.30656
Mitochondria selective S-nitrosation by mitochondria-targeted S-nitrosothiol protects against post-infarct heart failure in mouse hearts.
AIMS: Recently it has been shown that the mitochondria-targeted S-nitrosothiol MitoSNO protects against acute ischaemia/reperfusion (IR) injury by inhibiting the reactivation of mitochondrial complex I in the first minutes of reperfusion of ischaemic tissue, thereby preventing free radical formation that underlies IR injury. However, it remains unclear how this transient inhibition of mitochondrial complex I-mediated free radicals at reperfusion affects the long-term recovery of the heart following IR injury. Here we determined whether the acute protection by MitoSNO at reperfusion prevented the subsequent development of post-myocardial infarction heart failure. METHODS AND RESULTS: Mice were subjected to 30 min left coronary artery occlusion followed by reperfusion and recovery over 28 days. MitoSNO (100 ng/kg) was applied 5 min before the onset of reperfusion followed by 20 min infusion (1 ng/kg/min). Infarct size and cardiac function were measured by magnetic resonance imaging (MRI) 24 h after infarction. MitoSNO-treated mice exhibited reduced infarct size and preserved function. In addition, MitoSNO at reperfusion improved outcome measures 28 days post-IR, including preserved systolic function (63.7 ±1.8% LVEF vs. 53.7 ± 2.1% in controls, P = 0.01) and tissue fibrosis. CONCLUSIONS: MitoSNO action acutely at reperfusion reduces infarct size and protects from post-myocardial infarction heart failure. Therefore, targeted inhibition of mitochondrial complex I in the first minutes of reperfusion by MitoSNO is a rational therapeutic strategy for preventing subsequent heart failure in patients undergoing IR injury
MitoNeoD:a mitochondria-targeted superoxide probe
Mitochondrial superoxide (O2⋅−) underlies much oxidative damage and redox signaling. Fluorescent probes can detect O2⋅−, but are of limited applicability in vivo, while in cells their usefulness is constrained by side reactions and DNA intercalation. To overcome these limitations, we developed a dual-purpose mitochondrial O2⋅− probe, MitoNeoD, which can assess O2⋅− changes in vivo by mass spectrometry and in vitro by fluorescence. MitoNeoD comprises a O2⋅−-sensitive reduced phenanthridinium moiety modified to prevent DNA intercalation, as well as a carbon-deuterium bond to enhance its selectivity for O2⋅− over non-specific oxidation, and a triphenylphosphonium lipophilic cation moiety leading to the rapid accumulation within mitochondria. We demonstrated that MitoNeoD was a versatile and robust probe to assess changes in mitochondrial O2⋅− from isolated mitochondria to animal models, thus offering a way to examine the many roles of mitochondrial O2⋅−production in health and disease
Using exomarkers to assess mitochondrial reactive species in vivo
Background:
The ability to measure the concentrations of small damaging and signalling molecules such as reactive oxygen species (ROS) in vivo is essential to understanding their biological roles. While a range of methods can be applied to in vitro systems, measuring the levels and relative changes in reactive species in vivo is challenging.
Scope of review:
One approach towards achieving this goal is the use of exomarkers. In this, exogenous probe compounds are administered to the intact organism and are then transformed by the reactive molecules in vivo to produce a diagnostic exomarker. The exomarker and the precursor probe can be analysed ex vivo to infer the identity and amounts of the reactive species present in vivo. This is akin to the measurement of biomarkers produced by the interaction of reactive species with endogenous biomolecules.
Major conclusions and general significance:
Our laboratories have developed mitochondria-targeted probes that generate exomarkers that can be analysed ex vivo by mass spectrometry to assess levels of reactive species within mitochondria in vivo. We have used one of these compounds, MitoB, to infer the levels of mitochondrial hydrogen peroxide within flies and mice. Here we describe the development of MitoB and expand on this example to discuss how better probes and exomarkers can be developed. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.
Abbreviations:
EPR, electron paramagnetic resonance; GFP, green fluorescent protein; 4-HNE, 4-hydroxynonenal; MitoB, 3-(dihydroxyboronyl)benzyltriphenylphosphonium bromide; MitoP, (3-hydroxybenzyl)triphenylphosphonium bromide; ROS, reactive oxygen species; SOD, superoxide dismutase; TPMP, methyltriphenylphosphonium; TPP, triphenylphosphonium catio
Ischemic preconditioning protects against cardiac ischemia reperfusion injury without affecting succinate accumulation or oxidation.
Ischemia-reperfusion (IR) injury occurs when blood supply to an organ is disrupted and then restored, and underlies many disorders, notably myocardial infarction and stroke. While reperfusion of ischemic tissue is essential for survival, it also initiates cell death through generation of mitochondrial reactive oxygen species (ROS). Recent work has revealed a novel pathway underlying ROS production at reperfusion in vivo in which the accumulation of succinate during ischemia and its subsequent rapid oxidation at reperfusion drives ROS production at complex I by reverse electron transport (RET). Pharmacologically inhibiting ischemic succinate accumulation, or slowing succinate metabolism at reperfusion, have been shown to be cardioprotective against IR injury. Here, we determined whether ischemic preconditioning (IPC) contributes to cardioprotection by altering kinetics of succinate accumulation and oxidation during IR. Mice were subjected to a 30-minute occlusion of the left anterior descending coronary artery followed by reperfusion, with or without a protective IPC protocol prior to sustained ischemia. We found that IPC had no effect on ischemic succinate accumulation with both control and IPC mice having profound increases in succinate compared to normoxia. Furthermore, after only 1-minute reperfusion succinate was rapidly metabolised returning to near pre-ischemic levels in both groups. We conclude that IPC does not affect ischemic succinate accumulation, or its oxidation at reperfusion
Factors Predicting Long-term Outcome and the Need for Surgery in Graves Orbitopathy: Extended Follow-up From the CIRTED Trial
UNLABELLED: Graves Orbitopathy is both disabling and disfiguring. Medical therapies to reduce inflammation are widely used, but there is limited trial data beyond 18 months of follow-up. METHODS: 3 year follow-up of a subset of the CIRTED trial (N=68) which randomized patients to receive high dose oral steroid with azathioprine/placebo and radiotherapy/sham radiotherapy. RESULTS: Data were available at 3 years from 68 of 126 randomised subjects (54%). No additional benefit was seen at 3 years for patients randomized to azathioprine or radiotherapy with regard to a Binary Clinical Composite Outcome Measure, modified EUGOGO score or Ophthalmopathy Index.Clinical Activity Score (CAS), Ophthalmopathy Index and Total Eye Score improved over 3 years (p<0.001). However, quality of life at 3 years remained poor. Of 64 individuals with available surgical outcome data, 24/64 (37.5%) required surgical intervention. Disease duration of greater than 6 months before treatment was associated with increased need for surgery OR=16.8 (95%CI 2.95, 95.0) p=0.001. Higher baseline levels of CAS, Ophthalmopathy Index and Total Eye Score but not early improvement in CAS were associated with increased requirement for surgery. CONCLUSION: In this long-term follow-up from a clinical trial, 3 year outcomes remained suboptimal with ongoing poor quality of life and high numbers requiring surgery. Importantly, reduction in CAS in the first year, a commonly used surrogate outcome measure, was not associated with improved long-term outcomes
Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia.
Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO2--dependent S-nitrosation of proteins thiols in vivo Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO2- exposure
Leipziger und Schkeuditzer Gewässer: 24 Fließgewässer im Portrait
Der Auwald und seine Gewässer schlängeln sich wie grüne und blaue Bänder durch die Stadt Leipzig und ihre Umgebung
Metabolomic Profiling in Acute ST-Segment-Elevation Myocardial Infarction Identifies Succinate as an Early Marker of Human Ischemia-Reperfusion Injury.
BACKGROUND: Ischemia-reperfusion injury following ST-segment-elevation myocardial infarction (STEMI) is a leading determinant of clinical outcome. In experimental models of myocardial ischemia, succinate accumulation leading to mitochondrial dysfunction is a major cause of ischemia-reperfusion injury; however, the potential importance and specificity of myocardial succinate accumulation in human STEMI is unknown. We sought to identify the metabolites released from the heart in patients undergoing primary percutaneous coronary intervention for emergency treatment of STEMI. METHODS AND RESULTS: Blood samples were obtained from the coronary artery, coronary sinus, and peripheral vein in patients undergoing primary percutaneous coronary intervention for acute STEMI and in control patients undergoing nonemergency coronary angiography or percutaneous coronary intervention for stable angina or non-STEMI. Plasma metabolites were analyzed by targeted liquid chromatography and mass spectrometry. Metabolite levels for coronary artery, coronary sinus, and peripheral vein were compared to derive cardiac and systemic release ratios. In STEMI patients, cardiac magnetic resonance imaging was performed 2Â days and 6Â months after primary percutaneous coronary intervention to quantify acute myocardial edema and final infarct size, respectively. In total, 115 patients undergoing acute STEMI and 26 control patients were included. Succinate was the only metabolite significantly increased in coronary sinus blood compared with venous blood in STEMI patients, indicating cardiac release of succinate. STEMI patients had higher succinate concentrations in arterial, coronary sinus, and peripheral venous blood than patients with non-STEMI or stable angina. Furthermore, cardiac succinate release in STEMI correlated with the extent of acute myocardial injury, quantified by cardiac magnetic resonance imaging. CONCLUSION: Succinate release by the myocardium correlates with the extent of ischemia