Edit Abstract: Doerrier 2018 MiP2018a

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Abstract:	[[Image:MITOEAGLE-logo.jpg\|left\|100px\|link=http://www.mitoglobal.org/index.php/MITOEAGLE\|COST Action MitoEAGLE]] In recent years there has been an increasing interest in mitochondrial (mt) research in many physio-pathological conditions. In this context, the evaluation of mt-respiration is crucial for understanding mt-physiology and bioenergetics in health and disease. However, despite of the growing relevance in mt-research, there is no database which provides information about mt-function. [[MitoEAGLE network]] provides a powerful tool for establishing a useful mt-database in different species, tissues, and sample preparations. However, the comparison of reported data on mt-respiration from the scientific literature and inter/intra-laboratory comparisons are very difficult. In permeabilized muscle fibers (pfi) one of the critical issues comparing respiratory data is the quality of the mitochondrial preparations. Therefore, the generation of reference values which allow the evaluation of the skills preparing high-quality pfi, constitutes one of the main aims of the [[WG2]] (muscle) in the [[MitoEAGLE]] project. For this purpose, as a first step, we performed a pilot study collecting mt-respiratory data (pfi from mouse soleus) in different laboratories following the same experimental procedure. In this previous pilot study, we observed heterogeneous results between the groups involved [1]. In the present study, we addressed some potential aspects which could trigger this variability. Mitochondrial respiration was evaluated by [[high-resolution respirometry]] in pfi from soleus muscle from ''N''=15 C57BL6/J male mice using the [[SUIT-008 O2 pfi D014]] protocol (Fig. 1) [2]. Experiments were performed in parallel in the same laboratory by two researchers from different research groups. Our results did not exhibit significant differences on mitochondrial respiration by comparing individual mechanical sample preparations and, the effect of chemicals prepared for this study in both laboratories. On the other hand, we compared the results obtained for each researcher in the former and present study. We detected significant differences in oxygen consumption between both studies. Our findings demonstrate two sources of variability in mitochondrial respiration. First, ADP storage (time and/or temperature) seems to be a critical aspect affecting oxygen fluxes. Wet weight is the other important issue that must be considered and well-explained for generating reliable results in the future. The achievement of the reference values constitutes the starting point for obtaining reliable data in any project, avoiding an under/overestimation that could mask/influence relevant results in a study. This fact will contribute to decrease and maybe avoid non-reproducible data between different laboratories. Finally, this study will benefit the elaboration of a public mt-database in muscle permeabilized tissues.
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MiP areas:	Gender Respiration Instruments;methods mt-Biogenesis;mt-density mt-Structure;fission;fusion mt-Membrane mtDNA;mt-genetics nDNA;cell genetics Genetic knockout;overexpression Comparative MiP;environmental MiP Developmental biology Exercise physiology;nutrition;life style mt-Medicine Patients Pharmacology;toxicology mt-Awareness
Pathological states:	COPD Aging;senescence Alzheimer's Autism Cancer Cardiovascular Diabetes Inherited Infectious Myopathy Neurodegenerative Obesity Parkinson's Sepsis Other
Stress conditions:	Hypoxia Cell death Cryopreservation Ischemia-reperfusion Permeability transition Oxidative stress;RONS Temperature Mitochondrial disease
Organism:	Pig Human Mouse Rat Guinea pig Bovines Horse Dog Rabbit Cat Other mammals Chicken Zebrafish Drosophila Artemia Caenorhabditis elegans Saccharomyces cerevisiae Birds Reptiles Amphibians Fishes Chelicerates Crustaceans Hexapods Myriapods Annelids Molluscs Nematodes Other invertebrates Fungi Plants Protists Eubacteria Archea Algae
Tissues;cells:	Fat Heart Skeletal muscle Nervous system Liver Kidney Lung;gill Islet cell;pancreas;thymus Endothelial;epithelial;mesothelial cell Blood cells Genital Other cell lines CHO HEK HeLa Neuroblastoma Fibroblast HUVEC Macrophage-derived Lymphocyte Platelet Stem cells
Preparations:	SMP Intact organism Intact organ Permeabilized cells Permeabilized tissue Homogenate Isolated mitochondria Chloroplasts Enzyme Oxidase;biochemical oxidation Intact cells
Enzymes:	Complex I Adenine nucleotide translocase Complex II;succinate dehydrogenase Complex III Complex IV;cytochrome c oxidase Complex V;ATP synthase Inner mt-membrane transporter Marker enzyme Supercomplex TCA cycle and matrix dehydrogenases Uncoupling protein
Regulation:	pH Aerobic glycolysis ADP ATP ATP production AMP Calcium Coupling efficiency;uncoupling Cyt c Flux control Inhibitor Ion;substrate transport mt-Membrane potential Oxygen kinetics PCr;Cr Phosphate Redox state Substrate Temperature Threshold;excess capacity Uncoupler Fatty acid Amino acid Q-junction effect
Coupling:	ET LEAK ROUTINE OXPHOS
Pathways:	F N S Gp DQ CIV NS Other combinations ROX
High-resolution respirometry:	pH Oxygraph-2k TIP2k O2k-Fluorometer NO TPP Ca O2k-Spectrophotometer O2k-Manual O2k-Protocol Theory NextGen-O2k
Event:	A1 A2 A3 A4 B1 B2 B3 B4 C1 C2 C3 C4 D1 D2 D3 D4 E1 E2 Oral Poster P-flash Review
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Free text: Any additional information (e.g. affiliations of authors). == Affiliations and Support == :::: Doerrier C(1), Gama-Pérez P(2), García-Roves P(2,3), Gnaiger E(1,4) ::::# Oroboros Instruments, Innsbruck, Austria ::::# Dept Physiological Sciences, Univ Barcelona ::::# Bellvitge Biomedical Research Inst (IDIBELL); Spain ::::# Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. - carolina.doerrier@oroboros.at :::: Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 [[MitoEAGLE]]. == Figures == [[File:Doerrier_Figure_MiP2018.jpg|left|400 px]] Figure 1. Representative diagram for the [[SUIT-008 O2 pfi D014]] protocol. Briefly, 1PM, pyruvate and malate as substrates of NADH-linked pathway; 2D, saturated ADP to evaluate OXPHOS state in NADH-linked pathway; 2c, cytochrome c to assess the integrity of the outer mitochondrial membrane; 3G, glutamate for the NADH-pathway; 4S, succinate for NADH&Succinate pathway in OXPHOS state; 5U, uncoupler, for obtaining ET-state with NADH&Succinate linked-substrates; 6Rot, rotenone for inhibiting Complex I; 7Ama, antimycin A for blocking Complex III. == References == ::::#Garcia-Roves Pablo M, Chabi B, Doerrier C, Dubouchaud H, Grefte S, Irving B, Ost M, Pesta D (2017) Mitochondrial respirometry reference values from permeabilized mouse soleus muscle fibers. MitoEAGLE WG2 pilot study. [[Garcia-Roves_2017_MiP2017]] ::::#[[SUIT-008_O2_pfi_D014]]

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