Hoppel 2016c Abstract MitoFit Science Camp 2016

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Mitochondrial respiration in permeabilized skin fibroblasts.

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Hoppel CL (2016)

Event: MitoFit Science Camp 2016 Kuehtai AT

With Fang Ye, Ph.D., we have used cultured skin fibroblasts to provide a diagnostic step, before or as an alternative to an open muscle biopsy to examine systemic mitochondrial disorders [1].

Human skin fibroblasts were cultured in glucose containing medium and harvested to examine oxidative phosphorylation (OXPHOS) and to measure electron transfer-pathway (ET-pathway) complex activities. Assays were done using a high-resolution respirometer (Oroboros O2k), following two protocols. Protocol I, is similar to many studies [2], and measures oxygen consumption linked to electron transfer through Complex I, II, IV sequentially. Pyruvate and malate as the initial substrates as a tool to identify defects in pyruvate oxidation. The ADP-stimulated OXPHOS capacity was recorded after permeabilization by digitionin. Glutamate and succinate were sequentially added to obtain OXPHOS capacities with this substrate combination. An uncoupler, FCCP, then was added in a titration fashion to disassociate OXPHOS capacity from ET capacity, providing the maximum oxidative rate. Next, the addition of the Complex I inhibitor, rotenone allowed us to measure the Complex II-linked ET capacity. Antimycin A was then added to inhibit Complex III and obtain the residual oxygen consumption. Finally, the addition of TMPD and ascorbate provides the assessment of uncoupled Complex IV respiration, which is calculated as the azide–sensitive rate. In prototocol II, we assess fatty acid oxidation using palmitoylcarnitine and malate as substrates. Following rotenone addition, Complex III rate is measured by adding duroquinol and the ADP stimulated OXPHOS capacity is calculated. Finally, FCCP is titrated to acquire the noncoupled ET capacity; Antimycin A is used as the Complex III inhibitor.

We have obtained reproducible OXPHOS data from normal human skin fibroblast lines with multiple repeats and built reference intervals that indicate normal OXPHOS function. This approach passed the verification step with skin fibroblasts from a patient who had a Complex I defect that causes identifiable defects in skeletal muscle and isolated skeletal muscle mitochondria. The Complex I defect was observed in fibroblasts. We currently offer this approach for clinical diagnostics. Defects in mitochondrial oxidation and phosphorylation have been identified and possibly avert a muscle biopsy.


O2k-Network Lab: US OH Cleveland Hoppel CL


Labels: MiParea: Respiration, Patients 


Organism: Human  Tissue;cell: Endothelial;epithelial;mesothelial cell, Fibroblast  Preparation: Permeabilized cells 


Coupling state: OXPHOS, ET  Pathway: N, S, CIV, NS, ROX  HRR: Oxygraph-2k  Event: B2  MitoFit Science Camp 2016 

Affiliations

Center Mitochondr Diseases, Depts Pharmacology & Medicine, Case Western Reserve School Med, Cleveland, OH, USA. - charles.hoppel@case.edu

References

  1. Ye F, Hoppel CL (2013) Measuring oxidative phosphorylation in human skin fibroblasts. Anal Biochem 437:52-8. »Bioblast Link
  2. Lemieux H, Semsroth S, Antretter H, Höfer D, Gnaiger E (2011) Mitochondrial respiratory control and early defects of oxidative phosphorylation in the failing human heart. Int J Biochem Cell Biol 43:1729–38. »Bioblast Link