Stiles 2023 MiP2023

Stiles 2023 MiP2023

Mitochondrial respirometry and ATP hydrolysis measurements in previously frozen tissue samples

Link: MiP2023 Obergurgl AT

Stiles Linsey (2023)

Event: MiP2023 Obergurgl AT

Authors: Stiles Linsey, Fernandez-del-Rio L, Beninca C, Acin-Perez R, Shirihai Orian

Impaired mitochondrial function has been shown to play a key role in diseases of metabolism and aging. Respirometry is the gold standard measurement of mitochondrial function, as it is an integrated metabolic readout of the final step of the electron transport chain (ETC). However, analysis of mitochondrial respiratory function in tissue requires processing and measurement of freshly isolated mitochondria. This requirement makes respirometry impracticable for standard clinical practice, clinical studies, retrospective studies, and higher throughput respirometry. We have validated a methodology to measure maximal mitochondrial oxygen consumption rates through Complex I, II, and IV of the ETC in previously frozen biological samples using Agilent XF Analyzers. Additionally, Complex V (CV) ATP hydrolytic activity can be measured with the pH channel. These measurements of Complex I-V activities are specific as demonstrated by inhibition with ETC inhibitors. Additionally, these approaches can be applied to tissue homogenates, which simplifies the sample preparation and reduces the required starting material compared with isolating mitochondria. We find that primary changes in the maximal respiratory capacity, detected in fresh tissue, are preserved in frozen samples. These techniques to measure mitochondrial maximal respiratory function and CV hydrolytic activity in frozen samples makes clinical mitochondrial assessment more feasible and adds a complementary approach to investigate the role of mitochondrial function in disease onset and progression.

Affiliations

Stiles L^1,2, Fernández-del-Río L¹, Benincá C¹, Acín-Pérez R¹, and Shirihai O^1,2,3

1. Department of Medicine, Endocrinology, David Geffen School of Medicine, University of California, Los Angeles, CA, USA
2. Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA, USA
3. Molecular Biology Institute, UCLA, Los Angeles, CA, USA

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