# The effect of rotenone contamination on high-resolution mitochondrial respiration experiments

## TL;DR

This study shows that improper washing between experiments can lead to contamination by rotenone, affecting mitochondrial respiration measurements in high-resolution respirometry.

## Contribution

The study identifies a critical methodological flaw in high-resolution respirometry due to rotenone contamination and evaluates washing protocols to address it.

## Key findings

- The currently recommended wash protocol fails to remove rotenone effectively.
- Mitochondrial respiration decreased after exercise only in chambers exposed to rotenone between experiments.
- Inhibitor contamination can alter experimental conclusions in high-resolution respirometry.

## Abstract

High-resolution respirometry is commonly used in skeletal muscle research and exercise science to measure mitochondrial respiratory function in both permeabilized muscle fibers and isolated mitochondria. Due to the low throughput and high cost of the most used respirometer, the Oroboros 2k (O2k), multiple experiments are often conducted within the same chamber in short succession. Despite this, no methodological consideration has been given for the potential contamination of inhibitors, used to investigate the contribution of specific complexes within the electron transport chain, between experiments.

We first assessed the potential effect of inhibitor contamination on mitochondrial respiration experiments by evaluating the ability of the currently recommended wash protocol to remove rotenone and compared its efficacy against a simplified wash protocol of sequential rinses. Secondly, we assessed the potential effect of inhibitor contamination on mitochondrial respiration measured before and after a single session of high-intensity interval exercise, with and without the use of rotenone between experiments.

The currently recommended protocol for washing chambers was insufficient for removing rotenone. Following exercise, a decrease in mitochondrial respiration was observed exclusively in chambers exposed to rotenone between experiments.

Our findings highlight an important methodological consideration regarding the measurement of mitochondrial respiratory function using high-resolution respirometry, with inhibitor contamination potentially affecting the conclusions derived from experiments conducted in close succession. Future studies investigating mitochondrial respiratory function should assess the necessity of using inhibitors such as rotenone, ensure thorough wash procedures between experiments, and explicitly report the washing protocols used.