Chicco 2016a Abstract MitoFit Science Camp 2016
|Oxygen dependence of H2O2 release from skeletal muscle and cardiac mitochondria: influence of sample preparation.|
The development of the Oroboros O2k Fluorometry module enables researchers to simultaneously monitor oxygen consumption and H2O2 production from a single biological sample under the identical conditions over time. This facilitates direct assessment of mitochondrial H2O2 released per unit oxygen consumed, which is an important index of mitochondrial function relevant to studies of metabolic health and disease. While rigorous testing and validation of this instrument with different respiration media have been performed and published by the Oroboros team [1,2], experimental quality control and data interpretation remains the responsibility of the investigator.
Given well established oxygen diffusion limitation of respiration in some sample types at lower dissolved oxygen concentrations [H2O2] (e.g., permeabilized muscle fibers), we sought to evaluate the extent of these limitations on H2O2 flux in sample preparations commonly used to evaluate mitochondrial function in the literature. Using an Oroboros Oxygraph-2k and fluorometry module (Innsbruck, AT), we investigated the relationships between [O2] (0-400 μM), H2O2 release (Amplex Red), and oxygen flux during Complex I+II-linked ADP-phosphorylating respiration of isolated mitochondria, saponin-permeabilized fibers, and tissue homogenates obtained from skeletal muscle and cardiac tissue of C57Bl/6J mice.
As previously documented, our studies revealed a strong [O2] dependence of respiration in permeabilized fibers below ~250 μM that was not observed in isolated mitochondria or tissue homogenates. Conversely, H2O2release was strongly influenced by [O2] in isolated mitochondria and heart homogenates compared to muscle homogenates and permeabilized fibers from both tissues. Consequently, calculated H2O2released per O2 consumed tended to increase with decreasing [O2] below 250 μM in fibers, but steadily increased as [O2] decreased in mitochondria and homogenates from the same animals.
These results highlight the following points for consideration in the context of future respirometry protocol standardization and reporting:
a) Careful attention to [O2] during mitochondrial H2O2 flux experiments using this system is required to avoid erroneous and inconsistent results, particularly when expressing data relative to O2 flux and comparing data from different sample preparations b) H2O2 release from isolated mitochondria and homogenates is highly influenced by [O2] across all tensions studied, therefore, selecting a narrow range of [O2] where H2O2 flux is reported is suggested c) Permeabilized fibers may be preferred sample preparation for assessment of H2O2/O2 consumed, due to relatively stability of both measurements above 250 μM O2.
• O2k-Network Lab: US CO Fort Collins Chicco AJ
Labels: MiParea: Respiration
Organism: Mouse Tissue;cell: Heart, Skeletal muscle Preparation: Permeabilized tissue, Homogenate, Isolated mitochondria
Coupling state: OXPHOS Pathway: NS HRR: Oxygraph-2k, O2k-Fluorometer Event: C1 MitoFit Science Camp 2016, Amplex Red
Dept Biomedical Sci,Dept Biochemistry and Mol Biol, Colorado State Univ, CO, USA. - email@example.com
- Makrecka-Kuka M, Krumschnabel G, Gnaiger E (2015) High-resolution respirometry for simultaneous measurement of oxygen and hydrogen peroxide fluxes in permeabilized cells, tissue homogenate and isolated mitochondria. Biomolecules 5:1319-38. - »Bioblast link«
- Krumschnabel G, Fontana-Ayoub M, Sumbalova Z, Heidler J, Gauper K, Fasching M, Gnaiger E (2015) Simultaneous high-resolution measurement of mitochondrial respiration and hydrogen peroxide production. Methods Mol Biol 1264:245-61. - »Bioblast link«