Bezuidenhout 2016a Abstract MitoFit Science Camp 2016

Comparison of oxygen dependence of respiration in permeabilized mouse skeletal muscle fibers in two respiration media, MiR06Cr and Buffer Z containing Ctl, Cr and Blebbistatin.

Link:

Bezuidenhout N, Doerrier C, Droescher S, Ojuka E, Gnaiger E (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

It has been reported that the oxygen (O₂) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria [1]. Pfi are sensitive to low oxygen supply due to diffusion restrictions that limit the supply of oxygen to the core of the fiber bundle. Therefore, hyperoxic conditions (oxygen range 400-250 μM) have been employed to counteract this limitation. Further studies have shown that the addition of a myosin II-specific inhibitor, blebbistatin (BLEB) in the respiration medium reduces this sensitivity and allows the study of ADP kinetics in Pfi at normoxic oxygen levels (250-200 μM). Moreover, it has been described that the use of BLEB prevents fiber contraction and yields high K_m values for ADP [2].

In the present study, high-resolution respirometry (HRR) was used to test ADP respiratory kinetics in skeletal muscle Pfi from wild-type C57BL/6 mice under three different oxygen regimens: hypoxia (100-80 μM), normoxia (250-200 μM) and hyperoxia (400 μM) comparing two respiration media: Buffer Z with catalase (Ctl), creatine (Cr) and BLEB (BufferZCtlCr+BLEB) and MiR06+Cr (MiR06Cr) at a physiological temperature of 37 °C. Data were normalized to wet mass (Ww) (mg of tissue), Complex IV (COX) and citrate synthase (CS) activities.

In our experiments, we found no differences in O₂ flux when comparing rates determined in Buffer ZCtlCr + BLEB and MiR06Cr. However, our results do show that different O₂ regimens such as hypoxic and normoxic conditions in the O2k-chamber may lead to a relevant underestimation of OXPHOS capacity due to the oxygen limitation in both media (BufferZCtlCr+BLEB and MiR06Cr). It would therefore appear that, in our hands, the use of BufferZCtlCr with the addition of BLEB does not alleviate O₂ dependency in permeabilized skeletal muscle fibers.

• O2k-Network Lab: ZA Cape Town Smith J, AT Innsbruck Oroboros, ZA Cape Town Ojuka EO

Affiliations

1-Univ Cape Town, Dept Human Biol, South Africa; 2-Oroboros Instruments, Innsbruck, Austria; 3-D.Swarovski Research Lab, Dept Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck, Austria. - [email protected]

Figures

References and Support

Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25. »Bioblast link
Perry CG, Kane DA, Lin CT, Kozy R, Cathey BL, Lark DS, Kane CL, Brophy PM, Gavin TP, Anderson EJ, Neufer PD (2011) Inhibiting myosin-ATPase reveals a dynamic range of mitochondrial respiratory control in skeletal muscle. Biochem J 437:215-22. »Bioblast link

The project was supported by the K-Regio project K-Regio MitoFit, funded in part by the Tyrolian Government and the European Regional Development Fund (ERDF).

Publication

Doerrier C, Gama-Perez P, Pesta D, Distefano G, Soendergaard SD, Maise Chroeis K, Gonzalez-Franquesa A, Goodpaster BH, Prats C, Sales-Pardo M, Guimera R, Coen PM, Gnaiger E, Larsen S, Garcia-Roves PM (2024) Harmonization of experimental procedures to assess mitochondrial respiration in human permeabilized skeletal muscle fibers. Free Radic Biol Med 223:384-97. https://doi.org/10.1016/j.freeradbiomed.2024.07.039 - »Bioblast link«

Labels: MiParea: Respiration

Stress:Oxidative stress;RONS Organism: Mouse Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue

Regulation: ADP, Oxygen kinetics

HRR: Oxygraph-2k Event: C1 MitoFit Science Camp 2016, Blebbistatin