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

Participants

Participants were provided with information regarding the requirements of these studies (Fig. 1), including the benefits and risks, before providing both verbal and written informed consent. All participants were free of musculoskeletal injuries, any condition or disease that affects cardiovascular function (e.g., heart rhythm disturbance, elevated blood pressure, diabetes), and were not taking certain prescription medications (e.g., beta-blockers, anti-arrhythmic drugs, statins, or insulin-sensitizing drugs).

Experiments 1 & 2: Muscle biopsies were obtained at rest from 12 healthy men and 4 healthy women (30.8 ± 6.1 y; 174.0 ± 10.3 cm; 76.6 ± 16.6 kg, mean ± SD) from several studies approved by the Human Research Ethics Committee at Victoria University (HRE22-057, HRE20-065, and HRE22-184).

Experiment 3: Muscle biopsies were obtained from five healthy men and women (26.3 ± 4.5 y; 167.8 ± 4.7 cm; 74.4 ± 15.8 kg, mean ± SD) from a study approved by the Human Research Ethics Committee at Victoria University (HRE20-212).

Experiment 4: Muscle biopsies were obtained from 10 healthy men from a study approved by the Human Research Ethics Committee at Victoria University (HRE18-214). A full description of the recruitment and participant details has previously been described (Li et al., 2022).

Muscle biopsies

All participants reported to the laboratory in a fasted and rested state, having not had food, alcohol, or completed exercise in the 12 h prior to the biopsy trials. Following the injection of local anesthesia (1% lidocaine), an experienced medical doctor took all muscle biopsies from the vastus lateralis muscle using a suction-modified Bergstrom needle (Tarnopolsky et al., 2011). From each biopsy, 15 to 20 mg of muscle was rapidly placed into an ice-cold biopsy preservation solution (BIOPS) for high-resolution mitochondrial respiration. For Experiments 3 and 4, excess muscle was immediately frozen in liquid nitrogen and stored for the measurement of citrate synthase activity (as a marker of mitochondrial content; (Larsen et al., 2012)).

Exercise protocols

Both exercise trials (Experiments 3 and 4) used high-intensity interval exercise (HIIE) on a stationary bicycle. Similar HIIE protocols have been employed within 4- to 6-week training studies by our research group to significantly increase mitochondrial respiratory function (Granata, Jamnick & Bishop, 2018; Granata et al., 2016a).

Experiment 3: Following the first biopsy, the participants performed an exercise protocol consisting of four 4-min intervals, separated by 2 min of rest on a stationary bicycle (Velotron Cycle Ergometer; RacerMate Inc., Seattle, WA, USA). The intensity of each interval was set at the power at the lactate threshold (${\dot{W}}_{\mathrm{LT}}$) + 0.45 * (maximum power output (${\dot{W}}_{\max }$) –${\dot{W}}_{\mathrm{LT}}$), with ${\dot{W}}_{\max }$ and ${\dot{W}}_{\mathrm{LT}}$ determined using a ramped and graded exercise test, respectively, as previously performed by our group (Jamnick et al., 2018).

The second biopsy was taken 6 h following the completion of exercise (POST); this time point was selected due to the required time to complete both the pre-existing mitochondrial respiration SUIT protocol and the recommended Oroboros cleaning protocol (Di Marcello et al., 2023). Between biopsies, participants consumed a standardized diet of 2.2 g kg⁻¹ of bodyweight of carbohydrates, 0.7 g kg⁻¹ of bodyweight of protein, and 0.4 g kg⁻¹ of bodyweight of fat, which was spread across two meals eaten at approximately 0.5 and 3.5 h post exercise.

Experiment 4: Following the first biopsy, the participants performed an exercise protocol consisting of six 4-min intervals, separated by 2 min of rest, on a stationary bicycle (Velotron Cycle Ergometer; RacerMate Inc.). The intensity of each interval was set at 0.5 * peak power output (${\dot{W}}_{\mathrm{peak}}$) + 0.5 * ${\dot{W}}_{\mathrm{LT}}$, with ${\dot{W}}_{\mathrm{LT}}$ and ${\dot{W}}_{\mathrm{peak}}$ determined using a graded exercise test as previously performed by our group (Jamnick et al., 2018). A second biopsy was taken 3 h following the completion of exercise (POST). A full description of the baseline testing, exercise protocol, and the between-biopsy conditions have previously been described (Li et al., 2022).

Mitochondrial respiration

SUIT protocols

Experiment 1: Prior to analyzing the permeabilized and separated muscle fibers, ROT (final concentration in the chamber of 0.5 µM) was titrated into fully operational chambers containing MiR05. Cleaning using the simplified protocol was performed when ROT had been within the chamber for 15 min, consistent with its use within a typical SUIT protocol (Di Marcello et al., 2023).

The SUIT protocol used with final chamber concentration in brackets was as follows: pyruvate (two mM) and malate (five mM) in the absence of adenylates were added for measurement of leak respiration with electron entry through Complex I (CI_L) (oxygen consumption through Complex I with substrates present but no ADP). A series of stepwise titrations of ADP (100, 250, 500, 750, 1,000, 2,000, 5,000, 7,500, 10,000 µM) was then added and the final concentration served as a measurement of peak oxidative phosphorylation (OXPHOS) respiratory function with electron input through complex I (CI_P) (oxygen consumption through Complex I with substrates and ADP present to allow oxidative phosphorylation). Following this, succinate (10 mM) was added for the measurement of OXPHOS activity with simultaneous electron supply through Complex I and complex II (CI+II_P) (oxygen consumption through Complex I and Complex II with substrates and ADP present to allow oxidative phosphorylation). Cytochrome c (10 µM) was then added to test for outer mitochondrial membrane integrity; an exclusion criterion was set such that if a chamber showed an increase in O₂ flux over 15% after the addition of cytochrome c it was to be discarded from analysis, this threshold was based on previous assessments into mitochondrial respiration variability (Kuang et al., 2022). This was followed by stepwise carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone titrations (FCCP, 0.5–1 µM) for measurement of electron transport system (ETS) capacity with convergent electron input through complex I and II (CI+II_E) (oxygen consumption without the limitations of ATP synthase or membrane potential of CI+II_P). This was followed by the addition of antimycin A (AMA, 2.5 µM), an inhibitor of complex III, to obtain a measurement of residual oxygen consumption that was subtracted from all other measurements to account for non-respiration oxygen consumption in the chamber. A summary of SUIT protocols for all experiments is provided in Fig. 2.

Experiment 2: The same SUIT protocol as Experiment 1 was performed. However, instead of the simplified wash protocol following the initial titration of ROT, the recommended Oroboros wash protocol was utilized.

Experiment 3: The same SUIT protocol as Experiment 1 & 2 was performed; however, no ROT was titrated prior to the initial (PRE) biopsy. Instead, at the end of the SUIT protocol for the PRE samples, ROT (final concentration in the chamber of 0.5 µM) was titrated into half of the technical replicates. Cleaning using the recommended Oroboros protocol was performed when ROT had been within the chamber for at least 15 min (Di Marcello et al., 2023).

Experiment 4: The SUIT protocol used with final chamber concentration in brackets was as follows: octanoyl-carnitine (0.2 mM) and malate (two mM) in the absence of ADP was added to measure the leak respiration via electron transferring flavoprotein (ETF_L). A titration of MgCl₂ (three mM), ADP (5 mM), and pyruvate (five mM) were then added for measurement of respiration through ETF and complex I (ETF+CI_P). Followed by the addition of succinate (10 mM) for a measurement of peak OXPHOS respiratory function with simultaneous electron supply through ETF, complex I, and complex II (ETF + CI + II_P). cytochrome c (10 mM) was then added to check the integrity of mitochondrial outer membrane, followed by stepwise titrations carbonyl cyanide 4-(trifluoromethoxy) phenyl- hydrazone (FCCP) titrations to plateau (0.75–1.5 µM), for measurement of ETS capacity (ETF+CI+II_E) followed. Antimycin A (2.5 mM) was then added to measure residual oxygen consumption. Cleaning of the chambers between experiments occurred using the recommended Oroboros wash protocol.

Citrate synthase activity assay

Citrate synthase (CS) activity was determined using small sections of frozen skeletal muscle homogenized to a concentration of six µg µL⁻¹ for Experiment 3 and 4 µg µL⁻¹ for Experiment 4 in ice-cold lysis buffer containing 50 mM Tris, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM EDTA, and 1:100 (v/v) protease inhibitor cocktail (Cell Signaling Technology, Danvers, MA, USA) at a pH of 7.4. In triplicates, five µL of muscle homogenate was analyzed using 40 µL of three mM acetyl-CoA, 25 µL of one mM 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), and 165 µL 100 mM Tris buffer (pH 8.3) kept at 30 °C. After the addition of 15 µL of 10 mM oxaloacetic acid, the plate was quickly placed in an xMark Microplate spectrophotometer (Bio-Rad, Hercules, CA, USA), and after 30 s of linear agitation, absorbance at 412 nm was recorded every 15 s for 3 min at 30 °C.

Statistical analyses

The stats and emmeans packages in R were used for all statistical analyses, with the assumptions of a normal distribution and equal variance. A two-tailed paired student’s t-test was used to assess the effect of rotenone on mitochondrial respiration in Experiments 1 and 2, and the effect of exercise in Experiment 4. A repeated measures two-way ANOVA was used to test the effect of exercise, as well as the interaction between exercise and rotenone in Experiment 3. Post-hoc pairwise comparisons between groups in Experiment 3 were performed using estimated marginal means without adjustment for multiple comparisons, due to the small sample size, which limited the power of more stringent correction methods. Plots were generated using the ggplot2 package in R. Source data to verify this research is provided with this paper and the R script used for analysis is deposited on GitHub and available at https://doi.org/10.5281/zenodo.14862623. Significance was determined as a p-value < 0.05, and exact p-values were reported throughout. All values of mitochondrial respiration reported in the text are the mean difference between conditions or time points unless otherwise stated.

Rinsing does not completely remove rotenone following an experiment

The aim of Experiment 1 was to investigate whether a simplified wash, consisting of distilled H₂O, 70% (v/v) ethanol, and 100% (v/v) ethanol, was sufficient to remove rotenone from the chambers of an Oxygraph-2k between experiments conducted on permeabilized skeletal muscle fibers. There was no effect of ROT exposure on CI_L (p = 0.160; Fig. 3A). Measurements of CI_P (−61.13 pmol O2 s⁻¹ mg of wet weight⁻¹, p = 0.001; Fig. 3B), CI+II_P (−24.96 pmol O2 s⁻¹ mg of wet weight⁻¹, p = 0.037; Fig. 3C), and CI+II_E (−34.90 pmol O2 s⁻¹ mg of wet weight⁻¹, p = 0.013; Fig. 3D) were significantly lower in chambers that were exposed to ROT compared to those that were not (n = 6).

The currently recommended washing protocol is also insufficient at removing ROT contamination

Given that the simplified protocol was insufficient, we next investigated in Experiment 2 whether the currently recommended Oroboros wash protocol can remove rotenone from the chambers of an Oxygraph-2k between successive experiments conducted on permeabilized skeletal muscle fibers. There was again no effect of ROT exposure on CI_L (p = 0.565; Fig. 4A). However, a significantly lower CI_P (−9.99 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.026, Fig. 4B), CI+CII_P (−11.07 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.048; Fig. 4C), and CI+CII_E (−11.98 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.047; Fig. 4D) was still detected for chambers that were exposed to ROT, compared to those that were not (n = 12).

A decrease in respiration following exercise is only observed in chambers exposed to ROT

Several studies have reported a 15–40% decrease in the mass-specific respiration of permeabilized muscle fibers following a single session of exercise (Layec et al., 2018; Lewis et al., 2021; Trewin et al., 2018); however, an assessment of the potential contribution of inhibitor contamination to these results has not occurred. The aim of Experiment 3 was to investigate whether insufficient removal of rotenone might contribute to a reported decrease in mass-specific respiration in permeabilized skeletal muscle fibers following a single session of HIIE. Mass-specific mitochondrial respiration was significantly lowered following exercise in permeabilized human skeletal muscle when chambers were exposed to ROT (n = 4), but not in those that were not (n = 4).

Once again, no changes in CI_Lwere observed following exercise, regardless of exposure to ROT (Fig. 5A). Although CI_P (−21.91 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.097; Fig. 5B) and (−21.81 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.076; Fig. 5C) decreased within chambers exposed to ROT, they were below the threshold of significance. Following exercise, CI+II_E (−19.60 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.046; Fig. 5D) was significantly decreased when chambers were previously exposed to ROT. In the chambers not exposed to ROT after the PRE sample, there was no significant decrease in CI_P, CI+II_P, and CI+II_E following exercise (p = 0.452 to 0.967). No changes in CS activity, a commonly used indirect marker of mitochondrial content (Larsen et al., 2012), were detected following exercise (Fig. 5F).

Due to the relatively small sample size used within Experiment 3, additional data generated within our research group using a similar experimental design, but without the use of rotenone, were used to further assess if a single session of high-intensity interval exercise is associated with a significant decrease in mass-specific respiration in the absence of ROT (Experiment 4). There was no significant effect of exercise on mass-specific mitochondrial respiration measurements ETF+CI_L (p = 0.172; Fig. 6A), ETF+CI_P (−7.41 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.209; Fig. 6B), ETF+CI+II_P (−10.06 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.257; Fig. 6C), ETF+CI+II_E (−9.58 pmol O₂ s⁻¹ mg of wet weight⁻¹, p = 0.316; Fig. 6D) (n = 18). Like Experiment 3, no changes in CS activity were detected following exercise (Fig. 6F).