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), plasma membranes are either permeabilized or mitochondria are mechanically separated from tissues in mt-preparations. Permeabilized muscle fibres represent excellent mt-preparations, but require incubation at artificially high oxygen levels to overcome oxygen diffusion limitations [1]. Owing to large 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 ROS production and respiration. 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 and species.

In the present study with high-resolution respirometry (HRR), mt-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, making trout heart in this respect a better mt-model for human mt-cardiac function [4] than mouse heart. 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 compared to permeabilized fibres.

• 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

Extended abstract

In mouse heart homogenate, oxygen consumption and hydrogen peroxide production were monitored simultaneously by the modular extension of the OROBOROS Oxygraph-2k with the O2k-Fluorescence LED2-Module and application of Amplex Ultrared using minimum amounts of tissue (2 mg wet weight per chamber). While oxygen consumption was significantly less dependent on oxygen pressure in homogenate compared to permeabilized fibres, H2O2 production showed a reversible dependence on oxygen concentration that exceeded by far the effects of various substrate and coupling control states on the rate of hydrogen peroxide formation, in striking contrast to mouse brain mitochondria [5].

The remarkable species- and tissue-specific diversity of OXPHOS (substrate and coupling) control patterns will be discussed in relation to selecting appropriate models for comparative mitochondrial physiology and pathology, and for a variety of O2k-MultiSensor protocols applied in the functional diagnosis of mitochondrial performance. The fast and reproducible mt-preparation in tissue homogenates opens up new perspectives for comparative mitochondrial physiology.

Affiliations, acknowledgements and author contributions

OROBOROS INSTRUMENTS, Schöpfstr. 18, Innsbruck, Austria;

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

Email: erich.gnaiger@oroboros.at

Supported by K-Regio project MitoCom Tyrol.

References