scholarly journals Measuring flavin mononucleotide concentrations in whole blood, red cell based or acellular perfusate samples by fluorescence spectrometry

2021 ◽  
Author(s):  
Tine Wylin ◽  
Veerle Heedfeld ◽  
Ina Jochmans
Keyword(s):  
Whole Blood ◽  
Complex I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Internal Standard ◽  
Ex Situ ◽  
Fluorescence Spectrometry ◽  
Flavin Mononucleotide ◽  
Red Cell ◽  
Organ Perfusion

Abstract Flavin mononucleotide (FMN) is non-covalently bound to complex I of the mitochondrial respiratory chain. During ischemia-reperfusion injury, reduced FMN is released from complex I, leaving the complex impaired. In literature, FMN measured during ex situ organ perfusion has been considered as a biomarker for organ graft quality. With this protocol the FMN concentrations in perfusate samples taken during organ perfusion can be estimated quickly and easily by fluorescence spectrometry. The use of non-binding plates is essential. A fresh standard curve using riboflavin 5'-monophosphate sodium salt hydrate must be prepared every day. To quantitatively compare sample concentrations between different plates, it is advisable to include an internal standard. This internal standard is best prepared by pooling samples from experiments that use the same perfusate composition. This assay will work for samples acquired from whole blood, red cell based solutions or acellular solutions.

Abstract 1008: A Protective Role for Cardiac Specific Muscle Ring Finger-1 (MuRF1) in Ischemia/Reperfusion Injury In Vivo

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Monte S Willis ◽  
Mauricio Rojas ◽  
Pamela Lockyer ◽  
Thomas G Hampton ◽  
Luge Li ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Critical Role ◽  
Ring Finger ◽  
Left Ventricular ◽  
Ex Situ ◽  
Histological Evaluation ◽  
Area At Risk ◽  
Fractional Shortening

We previously identified a critical role for MuRF1 in suppressing pathologic cardiac hypertrophy. To extend these observations to other pathologic processes, we tested the role of MuRF1 in cardiac ischemia reperfusion (I/R) injury. We challenged MuRF1 transgenic (Tg) mice to I/R injury both ex situ and in vivo. First, we examined isolated MuRF1 Tg and age-matched sibling wild-type (WT) hearts after global ischemia (15 min) followed by reperfusion (20 min) in a Langendorff apparatus. Baseline function of MuRF1 Tg hearts did not significantly differ from WT hearts (mean left ventricular developed pressure (LVDP) 88.5 +/− 18 vs. 82.5 +/− 6.7, respectively; n = 4/group). Mean LVDP of hearts from MuRF1 Tg mice after reperfusion was 76.0 +/− 22.9% of baseline function compared to 27.2 +/− 13.3% in WT hearts (N = 5/group, P< 0.05)). To confirm that MuRF1 is cardioprotective in vivo, we subjected MuRF1 Tg and WT mice to a 30 minute ligation of the left anterior descending coronary artery, followed by 24 hours reperfusion. Mice underwent conscious echocardiography at baseline and after 24 hours; cardiac function was further interrogated by Millar pressure volume catheterization at 24 hours. Additionally, hearts underwent a histological evaluation of area at risk and infarct size. By echocardiography, a ~7% decrease in fractional shortening was identified in MuRF1 Tg mice after 24 hours reperfusion compared to baseline. This was in striking contrast to WT mice, which exhibited ~48% decrease in fractional shortening. Steady state catheterization measurements showed a significantly higher ejection fraction in MuRF1 Tg compared to WT mice after I/R injury (81.6 ± 2.3% vs. 49.0 +/− 4.0%, P < 0.05). Contractility reflected by +dP/dt max was better preserved in MuRF1 Tg compared to WT mice after I/R injury (12,614 +/− 776 vs. 7,448 +/−752, N = 3–12/group, P < 0.05). Histologically, the area of infarct in MuRF1 Tg mice was significantly smaller (10.0 +/− 0.8%) than in WT mice (25.5 +/− 2.5%, N = 4/group, P < 0.05). We demonstrate here for the first time that cardiac MuRF1 expression preserves function after I/R injury in vivo. Since MuRF1 is known to interact with metabolic and structural targets, this model will allow us to identify mechanisms by which MuRF1 modifies cardiac pathophysiology.

scholarly journals Nitrite attenuates mitochondrial impairment and vascular permeability induced by ischemia-reperfusion injury in the lung

2020 ◽  
Vol 318 (4) ◽  
pp. L580-L591
Author(s):  
Ajay Kumar ◽  
Kentaro Noda ◽  
Brian Philips ◽  
Murugesan Velayutham ◽  
Donna B. Stolz ◽  
...  
Keyword(s):  
Vascular Permeability ◽  
Complex I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Pulmonary Vascular Permeability ◽  
Ultrastructural Studies ◽  
Mitochondrial Impairment ◽  
Complex I Activity

Primary graft dysfunction (PGD) is directly related to ischemia-reperfusion (I/R) injury and a major obstacle in lung transplantation (LTx). Nitrite ([Formula: see text]), which is reduced in vivo to form nitric oxide (NO), has recently emerged as an intrinsic signaling molecule with a prominent role in cytoprotection against I/R injury. Using a murine model, we provide the evidence that nitrite mitigated I/R-induced injury by diminishing infiltration of immune cells in the alveolar space, reducing pulmonary edema, and improving pulmonary function. Ultrastructural studies support severe mitochondrial impairment in the lung undergoing I/R injury, which was significantly protected by nitrite treatment. Nitrite also abrogated the increased pulmonary vascular permeability caused by I/R. In vitro, hypoxia-reoxygenation (H/R) exacerbated cell death in lung epithelial and microvascular endothelial cells. This contributed to mitochondrial dysfunction as characterized by diminished complex I activity and mitochondrial membrane potential but increased mitochondrial reactive oxygen species (mtROS). Pretreatment of cells with nitrite robustly attenuated mtROS production through modulation of complex I activity. These findings illustrate a potential novel mechanism in which nitrite protects the lung against I/R injury by regulating mitochondrial bioenergetics and vascular permeability.

Subnormothermic isolated organ perfusion with Nicorandil increased cold ischemic tolerance of liver in experimental model

2021 ◽  
pp. 1-12
Author(s):  
Luca Erlitz ◽  
Caleb Ibitamuno ◽  
Benedek Kasza ◽  
Vivien Telek ◽  
Péter Hardi ◽  
...  
Keyword(s):  
Ischemia Reperfusion Injury ◽  
Ex Vivo ◽  
Histopathological Examination ◽  
Ischemia Reperfusion ◽  
Liver Perfusion ◽  
Ischemic Tolerance ◽  
Organ Perfusion ◽  
Hepatic Ischemia ◽  
Rat Liver Perfusion ◽  
Isolated Organ Perfusion

BACKGROUND: The cold ischemia –reperfusion injury may lead to microcirculatory disturbances, hepatocellular swelling, inflammation, and organ dysfunction. Nicorandil is an anti-ischemic, ATP-sensitive potassium (KATP) channel opener drug and has proved its effectiveness against hepatic Ischemia/Reperfusion (I/R) injury. OBJECTIVE: This study aimed to investigate the effect of Nicorandil on mitochondrial apoptosis, oxidative stress, inflammation, histopathological changes, and cold ischemic tolerance of the liver in an ex vivo experimental isolated-organ-perfusion model. METHODS: We used an ex vivo isolated rat liver perfusion system for this study. The grafts were retrieved from male Wistar rats (n = 5 in each), preserved in cold storage (CS) for 2 or 4 hours (group 1, 2), or perfused for 2 or 4 hours (group 3, 4) immediately after removal with Krebs Henseleit Buffer (KHB) solution or Nicorandil containing KHB solution under subnormothermic (22–25°C) conditions (group 5, 6). After 15 minutes incubation at room temperature, the livers were reperfused with acellular, oxygenated solution under normothermic condition for 60 minutes. RESULTS: In the Nicorandil perfused groups, significantly decreased liver enzymes, GLDH, TNF-alpha, and IL-1ß were measured from the perfusate. Antioxidant enzymactivity was higher in the perfused groups. Histopathological examination showed ameliorated tissue deterioration, preserved parenchymal structure, decreased apoptosis, and increased Bcl-2 activity in the Nicorandil perfused groups. CONCLUSIONS: Perfusion with Nicorandil containing KHB solution may increase cold ischemic tolerance of the liver via mitochondrial protection which can be a potential therapeutic target to improve graft survival during transplantation.

scholarly journals Electron leak from NDUFA13 within mitochondrial complex I attenuates ischemia-reperfusion injury via dimerized STAT3

2017 ◽  
Vol 114 (45) ◽  
pp. 11908-11913 ◽  
Author(s):  
Hengxun Hu ◽  
Jinliang Nan ◽  
Yong Sun ◽  
Dan Zhu ◽  
Changchen Xiao ◽  
...  
Keyword(s):  
Molecular Structure ◽  
Oxygen Consumption ◽  
Complex I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Ros Generation ◽  
Cell Protection ◽  
Causative Relationship ◽  
R Process ◽  
And Function

The causative relationship between specific mitochondrial molecular structure and reactive oxygen species (ROS) generation has attracted much attention. NDUFA13 is a newly identified accessory subunit of mitochondria complex I with a unique molecular structure and a location that is very close to the subunits of complex I of low electrochemical potentials. It has been reported that down-regulated NDUFA13 rendered tumor cells more resistant to apoptosis. Thus, this molecule might provide an ideal opportunity for us to investigate the profile of ROS generation and its role in cell protection against apoptosis. In the present study, we generated cardiac-specific tamoxifen-inducible NDUFA13 knockout mice and demonstrated that cardiac-specific heterozygous knockout (cHet) mice exhibited normal cardiac morphology and function in the basal state but were more resistant to apoptosis when exposed to ischemia-reperfusion (I/R) injury. cHet mice showed a preserved capacity of oxygen consumption rate by complex I and II, which can match the oxygen consumption driven by electron donors ofN,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD)+ascorbate. Interestingly, at basal state, cHet mice exhibited a higher H2O2level in the cytosol, but not in the mitochondria. Importantly, increased H2O2served as a second messenger and led to the STAT3 dimerization and, hence, activation of antiapoptotic signaling, which eventually significantly suppressed the superoxide burst and decreased the infarct size during the I/R process in cHet mice.

scholarly journals Mitochondrial Telomerase Reverse Transcriptase Protects from Myocardial Ischemia/reperfusion Injury by Improving Complex I Composition and Function

Circulation ◽  
2021 ◽  
Author(s):  
Niloofar Ale-Agha ◽  
Philipp Jakobs ◽  
Christine Goy ◽  
Mark Zurek ◽  
Julia Rosen ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ischemic Preconditioning ◽  
Mitochondrial Respiration ◽  
Complex I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Telomerase Reverse Transcriptase ◽  
Myofibroblast Differentiation ◽  
Migratory Capacity ◽  
Complex I Activity

Background: The catalytic subunit of telomerase, Telomerase Reverse Transcriptase (TERT) has protective functions in the cardiovascular system. TERT is not only present in the nucleus, but also in mitochondria. However, it is unclear whether nuclear or mitochondrial TERT is responsible for the observed protection and appropriate tools are missing to dissect this. Methods: We generated new mouse models containing TERT exclusively in the mitochondria (mitoTERT mice) or the nucleus (nucTERT mice) to finally distinguish between the functions of nuclear and mitochondrial TERT. Outcome after ischemia/reperfusion, mitochondrial respiration in the heart as well as cellular functions of cardiomyocytes, fibroblasts, and endothelial cells were determined. Results: All mice were phenotypically normal. While respiration was reduced in cardiac mitochondria from TERT-deficient and nucTERT mice, it was increased in mitoTERT animals. The latter also had smaller infarcts than wildtype mice, whereas nucTERT animals had larger infarcts. The decrease in ejection fraction after one, two and four weeks of reperfusion was attenuated in mitoTERT mice. Scar size was also reduced and vascularization increased. Mitochondrial TERT protected a cardiomyocyte cell line from apoptosis. Myofibroblast differentiation, which depends on complex I activity, was abrogated in TERT-deficient and nucTERT cardiac fibroblasts and completely restored in mitoTERT cells. In endothelial cells, mitochondrial TERT enhanced migratory capacity and activation of endothelial NO synthase. Mechanistically, mitochondrial TERT improved the ratio between complex I matrix arm and membrane subunits explaining the enhanced complex I activity. In human right atrial appendages, TERT was localized in mitochondria and there increased by remote ischemic preconditioning. The Telomerase activator, TA-65 evoked a similar effect in endothelial cells, thereby increasing their migratory capacity, and enhanced myofibroblast differentiation. Conclusions: Mitochondrial, but not nuclear TERT, is critical for mitochondrial respiration and during ischemia/reperfusion injury. Mitochondrial TERT improves complex I subunit composition. TERT is present in human heart mitochondria, and remote ischemic preconditioning increases its level in those organelles. TA-65 has comparable effects ex vivo and improves migratory capacity of endothelial cells and myofibroblast differentiation. We conclude that mitochondrial TERT is responsible for cardioprotection and its increase could serve as a therapeutic strategy.

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