A novel approach to measure mitochondrial respiration in frozen biological samples.

Rebeca Acín‐Pérez ,Sandrine Lagarrigue ,Arianne Caudal ,Marc Liesa ,Francesca Amati ,Harold S Sacks ,Ilan Y Benador ,Ajit S Divakaruni ,Georgios Karamanlidis ,Gloria A Benavides ,Linsey Stiles ,Rong Tian ,Jonathan Wanagat ,Michaela Veliova ,Anton Petcherski ,Anne N Murphy ,Laurent Vergnes ,Victor Darley‐Usmar ,Karen Reue ,Orian S Shirihai

doi:10.15252/embj.2019104073

Abstract

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. However, the requirement for freshly isolated mitochondria hinders the feasibility of respirometry in multi‐site clinical studies and retrospective studies. Here, we describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection. This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks.

Highlights

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together
Mitochondria (4 lg protein/well) isolated from fresh and frozen mouse livers were assayed in the presence of pyruvate and malate, as substrates, and sequential injections of ADP, oligomycin, FCCP, and antimycin A/rotenone followed. mFrozen assayed using pyruvate and malate as substrates showed significantly lower oxygen consumption rate (OCR) in all respiratory states compared to mitochondria isolated from fresh tissue (Fig 1A–C)
To control for non-specific respiration, we confirmed that succinatedependent mFrozen respiration is inhibited by the Complex IIspecific inhibitor 3-nitroproprionic acid in a concentration-dependent manner (Appendix Fig S1A). mFrozen pre-incubated with succinate showed a significantly higher respiratory rate than mFresh

Summary

Introduction

Respirometry is the gold standard measurement of mitochondrial oxidative function, as it reflects the activity of the electron transport chain complexes working together. We describe a novel respirometry approach suited for frozen samples by restoring electron transfer components lost during freeze/thaw and correcting for variable permeabilization of mitochondrial membranes. This approach preserves 90–95% of the maximal respiratory capacity in frozen samples and can be applied to isolated mitochondria, permeabilized cells, and tissue homogenates with high sensitivity. We find that primary changes in mitochondrial function, detected in fresh tissue, are preserved in frozen samples years after collection This approach will enable analysis of the integrated function of mitochondrial Complexes I to IV in one measurement, collected at remote sites or retrospectively in samples residing in tissue biobanks

Methods

Results

Conclusion