Krumschnabel 2013 Abstract MiP2013

Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for OXPHOS analysis in comparative mitochondrial physiology: trout and mouse – heart and liver mitochondria. Mitochondr Physiol Network 18.08.

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Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013)

Event: MiP2013

OXPHOS analysis is based on measurement of respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of various flux control variables, X (substrates, ADP, etc.), to the mitochondria (mt), the plasma membranes have to be either permeabilized or the mitochondria must be mechanically separated from the intact cell in mt-preparations. Permeabilized muscle fibres represent an excellent and gentle type of mt-preparation, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large oxygen diffusion gradients and the oxygen dependence of mt-H2O2 production over a wide range of oxygen pressure [2], permeabilized muscle fibres may not represent an adequate model for the combined study of respiration and ROS production. A high-quality preparation of tissue homogenate may represent an optimum compromise for a variety of respirometric and fluorometric studies. These considerations provided the rationale for initiating a study with the PBI-Shredder, an auxiliary HRR-Tool providing a standardized approach to prepare homogenates of various tissues (heart, liver, brain) and species (mouse, rainbow trout). In the present study with high-resolution respirometry, mitochondrial respiratory control was compared in trout heart and liver tissue homogenate preparations at 15 °C [3]. Biochemical coupling efficiency with Complex I (CI)-linked substrates were identical in the two tissues. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS in trout heart than liver. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate added to glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. For comparison, mouse heart and liver homogenate was measured at 37 °C using an identical substrate-uncoupler-inhibitor titration (SUIT) protocol. The cytochrome c test (<5% stimulation in healthy controls) indicated outer mt-membrane integrity in all cases, following an optimization of the PBI-Shredder application with high reproducibility of complete mt-yield and preservation of mitochondrial respiratory control.

• O2k-Network Lab: AT Innsbruck Gnaiger E, AT Innsbruck OROBOROS, AT Innsbruck MitoCom

Labels: MiParea: Respiration, Instruments;methods, Comparative MiP;environmental MiP 

Organism: Mouse  Tissue;cell: Heart, Nervous system, Liver  Preparation: Homogenate 

Regulation: Coupling efficiency;uncoupling, Cyt c  Coupling state: LEAK, OXPHOS, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Oxygraph-2k, Fluorometry"Fluorometry" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

MiP2013 

Affiliations, acknowledgements and author contributions

Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck;

OROBOROS INSTRUMENTS, Schöpfstr. 18, Innsbruck, Austria

Email: erich.gnaiger@oroboros.at

Supported by K-Regio project MitoCom Tyrol.

References

1. Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543: 39-55.
2. Boveris A, Chance B (1973) The mitochondrial generation of hydrogen peroxide. General properties and effect of hyperbaric oxygen. Biochem J 134: 707-716.
3. Doerrier CV, Draxl A, Wiethüchter A, Eigentler A, Gnaiger E (2013) Mitochondrial respiration in permeabilized fibres versus homogenate from fish liver and heart. An application study with the PBI-Shredder. Mitochondr Physiol Network 17.03 V3: 1-12.
4. Fasching M, Sumbalova Z, Gnaiger E (2013) O2k-Fluorometry: HRR and H2O2 production in mouse brain mitochondria. Mitochondr Physiol Network 17.03 V2: 1-4.