Doerrier 2014 Abstract SECF

From Bioblast
Jump to: navigation, search
Evaluation of melatonin treatment in mitochondria of permeabilized myocardial fibers from septic mice.

Link:

Doerrier C, Volt H, Garcia JA, Diaz-Casado ME, Lima-Cabello E, Escames G, Lopez LC, Acuna-Castroviejo D (2014)

Event: SECF

Sepsis, a systemic inflammatory response to infection, represents the major cause of mortality in the intensive care units of developed countries. Sepsis-induced reactive oxygen and nitrogen species (ROS/RNS) results in oxidative-nitrosative stress, which might finally lead to proteins, DNA, and lipid damage, causing severe mitochondrial dysfunction. Analysis of mitochondrial respiratory function in permeabilized fibers (Pfi) is essential for studying intracellular energy metabolism in physiological and pathological conditions. Pfi permit to maintain normal mitochondrial interactions and organization within cells compared to standard procedures, such as mitochondrial isolation. In addition, Pfi preserve appropriate properties of mitocondria, and a proper yield of mitochondrial content (≥ 95%) with representative functional and pathological mitochondria. The effects of melatonin (aMT), which prevents septic shock and multiple organ failure, have been related to the inhibition of iNOS expression, blunting the elevated levels of NO• and ROS, and restoring electron transport chain activity and ATP production. Therefore, we studied here whether Pfi are suitable to evaluate the effects of sepsis and melatonin treatment on mitochondrial function in Pfi.

Mitochondrial function was analyzed during experimental sepsis induced by cecal ligation and punture (CLP) in hearts of 3 mo. C57BL/6 mice. Melatonin treatment (30 mg/ml) was administered to mice at dose of 30 mg/kg body weight. Fig. 1 shows the experimental design and experimental groups. Mitochondrial respiration was assessed in permeabilized myocardial fibers in the presence of different substrate combinations by high resolution respirometry. Mitochondrial content of cytochromes was also analysed by spectrophotometry, and citrate synthase (CS) activity was measured as mitochondrial mass marker. Supramolecular organization of mitochondrial respiratory complexes as supercomplexes (SC) was determinate by Blue-Native gel electrophoresis.

OXPHOS capacities with complex I-linked substrates (glutamate+malate and glutamate+malate+pyruvate) showed a significant decay during sepsis. The physiological OXPHOS capacity (with complexes I+II substrate cocktail) was also strongly altered in experimental sepsis. OXPHOS capacities impairment was accompanied by a drop in electron transport system (ET-pathway) capacities (for complexes I+II- and complex IIrespiration). We observed a reduction in CS activity at 24 hours after septic induction. A decline in complex III incorporated into SC was also detected. No changes in cytochromes content were observed. Melatonin protected mitochondria, mantaining optimal bioenergetics and preventing changes in mitochondrial SC due to sepsis.

Keywords: sepsis, melatonin, mitochondrial bioenergetics

O2k-Network Lab: ES Granada Acuna-Castroviejo D


Labels: MiParea: Respiration, Pharmacology;toxicology  Pathology: Sepsis 

Organism: Mouse  Tissue;cell: Heart  Preparation: Permeabilized tissue, Isolated mitochondria  Enzyme: Marker enzyme, Supercomplex 

Coupling state: LEAK, OXPHOS, ET  Pathway: N, S, NS, ROX  HRR: Oxygraph-2k  Event: Oral 


Affiliations

1-Instituto de Biotecnología, Centro de Investigación Biomédica, Parque Tecnológico de Ciencias de la Salud, Universidad de Granada, Avda. Del Conocimiento s/n, 18100 Armilla, Granada, Spain. 2-Departamento de Fisiología, Facultad de Medicina,Universidad de Granada, Avenida de Madrid 11, 18012 Granada, Spain. 3-Unidad de Gestión Clínica de Laboratorios Clínicos, Hospital Universitario San Cecilio, Avenida Dr. Olóriz s/n, 18012 Granada, Spain.

Abstract continued

Our results evidence a loss of mitochondrial density during sepsis. Moreover, sepsis induced a severe bioenergetic impairment with all substrate combinations used. Melatonin preserved the respiratory supramolecular organization, maintaining their electron transport system capacity, and an adequate mitochondrial function. Thus, this work reinforces the utility of Pfi to detect mitochondrial impairment during sepsis and the efficacy of melatonin treatment to maintain mitochondrial homeostasis. The results also reinforce the potential clinical utility of melatonin in sepsis.

Acknowledgements

Supported by grants # PI13-00981