Krumschnabel 2013 Abstract MiP2013
|Krumschnabel G, Eigentler A, Fontana-Ayoub M, Draxl A, Fasching M, Gnaiger E (2013) Tissue homogenates for respiratory OXPHOS analysis in comparative mitochondrial physiology: mouse and trout – heart and liver. Mitochondr Physiol Network 18.08.|
OXPHOS analysis is based on measurement of mitochondrial (mt) respiration in various steady-states of substrate supply and coupling of electron transfer to phosphorylation of ADP. To secure full accessibility of any flux control variables (substrates, ADP, etc.) to the mt organelle, mitochondria are either isolated from their cellular containment or plasma membranes are mechanically or chemically permeabilized. However, while permeabilized muscle fibres represent excellent mt-preparations, they require incubation at artificially high oxygen levels to avoid oxygen diffusion limitation . On the other hand, mt-H2O2 production is oxygen dependent over a wide range of oxygen pressure . Uncontrolled oxygen gradients in permeabilized muscle fibres, therefore, argue against application of this model for the combined study of ROS production and respiration. A high-quality preparation of tissue homogenate could eliminate diffusion restrictions and thus the need for elevated oxygen levels. This may provide an optimum compromise for a variety of respirometric and fluorometric studies. Therefore, we evaluated the PBI-Shredder as an auxiliary HRR-tool for a standardized preparation of homogenates from various tissues and species. In the present study we applied high-resolution respirometry (HRR) to characterize and compare homogenate preparations from heart and liver of trout and mouse at the respective physiologically relevant temperature of 15 °C and 37 °C . In trout heart and liver biochemical coupling efficiency with Complex I (CI)-linked substrates was identical in the two tissues. CI-linked substrate control capacity (OXPHOS) was higher whereas CII-linked succinate control capacity was lower in heart than liver. Pyruvate enhanced glutamate+malate stimulated OXPHOS capacity to a larger extent in heart than liver. The ADP-ATP phosphorylation system exerted a higher control over OXPHOS (CI+II) in heart than liver, making trout heart in this respect a better mt-model for human mt-cardiac function  than mouse heart. Mouse heart and liver homogenates were 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.
Labels: MiParea: Respiration, Instruments;methods, Comparative MiP;environmental MiP Mammal;model: Mouse Non-mammal: Fishes Tissue;cell: Heart, Nervous system, Liver Preparation: Homogenate Regulation: Coupling efficiency;uncoupling, Cyt c Coupling state: LEAK, OXPHOS, ETS Substrate state: CI, CII, CI&II, ROX HRR: Oxygraph-2k, O2k-Fluorometer Additional: MiP2013, PBI-Shredder
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). The oxygen-independent range was significantly extended 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 .
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 for functional diagnosis of mitochondrial performance. The fast and reproducible mt-preparation in tissue homogenates opens up new perspectives for comparative mitochondrial physiology.
For the figures see pdf MiP2013 Book of Abstracts: pp 26-27
1 - OROBOROS INSTRUMENTS, Schöpfstr. 18, Innsbruck, Austria;
2 -Daniel Swarovski Research Laboratory, Department of Visceral, Transplant and Thoracic Surgery, Medical University of Innsbruck, Austria.
Supported by K-Regio project MitoCom Tyrol.
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