Hoppel 2016b Abstract MitoFit Science Camp 2016
|Comparison of permeabilized skeletal muscle fibers or isolated mitochondria for the detection of oxidative phosphorylation defects.|
We use the measurement of integrated mitochondrial function or oxidative phosphorylation (OXPHOS) to evaluate mitochondrial dysfunction in energy production as a diagnostic tool in skeletal muscle. During an one year period we used both isolated skeletal muscle mitochondria and permeabilized fibers from the same biopsy to evaluate OXPHOS capacity.
The goal of our study was to perform a quantitative and qualitative comparison of those two methods in muscle biopsy samples (vastus lateralis). The muscle sample from 40 patients was used for (1) isolation of mitochondria, and (2) muscle fibers permeabilization. Oxygen consumption was performed with the isolated mitochondria using a conventional oxygraph and standard protocols (performed by Mariana Rosca, M.D. ), while for the permeabilized fibers, high-resolution respirometry combined with a multiple substrate-uncoupler-inhibitor-titration (SUIT) protocol was used (Edwin Vazquez ). Citrate synthase activity (Hiral Patel), a marker of mitochondrial content, was compared in homogenate of muscle and permeabilized fibers removed from the chamber after the estimation of OXPHOS. In a group identified as “pathological controls”, a quantitative comparison of the OXPHOS capacities obtained was performed between the two methods (studies done by Helene Lemieux, Ph.D.). Oxygen flux in isolated mitochondria was expressed per g wet weight of muscle using the mitochondrial protein yield from the muscle, or per unit of citrate synthase activity. Furthermore, a factor was applied for the conversion of flux in isolated mitochondria measured at 30 °C to the corresponding flux in the permeabilized fibers measured at 37 °C . Independent of how the data are expressed, the estimation of mitochondrial respiration for substrates entering at Complexes I, II, or IV were similar if not identical in both methods. The careful and quantitative evaluation of methods available for measurement of mitochondrial function is important for combining such results in a comparative data base .
1-Center Mitochondr Diseases, Depts Pharmacology & Medicine, Case Western Reserve School Med, Cleveland, OH, USA. - [email protected]
2-Daniel Swarovski Research Laboratory, Dept General Transplant Surgery, Med Univ Innsbruck, Austria
- Puchowicz MA, Varnes ME, Cohen BH, Friedman NR, Kerr DS, Hoppel CL (2004) Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria-case studies. Mitochondrion 4:377-85. »Bioblast Link
- Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. »Bioblast Link
Labels: MiParea: Respiration, Patients
Organism: Human Tissue;cell: Skeletal muscle Preparation: Permeabilized cells, Homogenate, Isolated mitochondria
Pathway: N, S, CIV HRR: Oxygraph-2k Event: D1