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.|
It has been reported that the oxygen (O2) dependence of mitochondrial respiration in permeabilized fibers (Pfi) is ~100-fold higher compared to isolated mitochondria . 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 Km values for ADP .
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 weight (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.
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
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]
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).