Doerrier 2017 MiP2017 WG2
Oxygen dependence of mitochondrial respiration has been described in permeabilized fibers (pfi) . Therefore, high oxygen levels (400-250 µM) are required in pfi to avoid oxygen limitation. However, many laboratories use different conditions (e.g. protocols, media and oxygen regimes) for the evaluation of mitochondrial respiration in pfi. In the MitoEAGLE project, one of the major aims of WG2 is collecting muscle data from human and animal models around the world for a comparative mitochondrial physiology study. An important issue for this goal is the harmonization of protocols for high-resolution respirometry (HRR) with pfi.
In the present work, HRR was used to evaluate mitochondrial respiration in pfi from human (vastus lateralis, N=2) and C57BL6/J mice (soleus, N=3) biopsies in Buffer Z and MiR05-Kit (enriched with 20 mM of creatine) under different oxygen regimes. It has been reported that blebbistatin (a myosin II-specific inhibitor) allows the study of mitochondrial respiration in pfi at normoxic oxygen levels (220-150 μM) and avoids fiber contraction . Accordingly, in the present study 25 µM blebbistatin was incorporating to the Buffer Z and MiR05-Kit. A comparison between MiR05-Kit with and without blebbistatin was included. The NADH-pathway (N-pathway) was evaluated in the presence of 5 mM glutamate and 2 mM malate in LEAK and in OXPHOS states. After the evaluation of the outer mitochondrial membrane integrity by the addition of 10 µM cytochrome c, the combined NADH and succinate-pathway (NS; 10 mM succinate) was investigated in different oxygen regime: normoxia (240-170 µM) and hyperoxia (450-250 µM). Finally, NS-pathway capacity was obtained by titrating the uncoupler FCCP. Oxygen was maintained in the optimal levels by titrating H2O2 in the presence of catalase (280 U/mL).
In our preliminary study, we did not find significant differences in oxygen flux normalized by wet weight between Buffer Z and MiR05-Kit (with and without blebbistatin). A similar 7-10% oxygen dependence was observed in human vastus lateralis in the two media, whereas it was more pronounced and more heterogeneous in soleus tissue from mouse (14%±11 SD in Buffer Z with blebbistatin, 18%±6 SD in MiR05-Kit with blebbistatin and 28%±24 SD in MiR05-Kit without blebbistatin). These results agree with previous experiments, where we observed no differences between MiR05 and Buffer Z media and similar behavior in the oxygen dependence . However, further experiments must be performed in order to investigate properly the effects of oxygen and ADP on OXPHOS capacity in pfi using different media in the presence and absence of blebbistatin.
Labels: MiParea: Respiration, Pharmacology;toxicology
Organism: Mouse Tissue;cell: Skeletal muscle Preparation: Permeabilized tissue
Regulation: ADP, Inhibitor, Oxygen kinetics Coupling state: LEAK, OXPHOS, ET Pathway: N, NS
Affiliations and support
- Doerrier C(1), Distefano G(2), Coen P(2), Goodpaster B(2), Gnaiger E(1,3)
- Oroboros Instruments, Innsbruck, Austria
- Translational Research Inst Metabolism Diabetes, Florida Hospital, Orlando, USA
- Dept Visceral, Transplant Thoracic Surgery, Daniel Swarovski Research Lab, Medical Univ Innsbruck, Austria. - firstname.lastname@example.org
- Contribution to European Union Framework Programme Horizon 2020 COST Action CA15203 MitoEAGLE.
- Scandurra FM, Gnaiger E (2010) Cell respiration under hypoxia: facts and artefacts in mitochondrial oxygen kinetics. Adv Exp Med Biol 662:7-25.
- 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.
- Bezuidenhout N, Doerrier C, Droescher S, Ojuka E, Gnaiger E (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. Abstract MitoFit Science Camp 2016.