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. Author manuscript; available in PMC: 2026 Sep 11.
Published in final edited form as: Biochim Biophys Acta Bioenerg. 2025 Aug 11;1866(4):149568. doi: 10.1016/j.bbabio.2025.149568

Chamber oxygen concentration impacts mitochondrial function and hydrogen peroxide appearance in permeabilized human skeletal muscle fibers

Bradley A Ruple a,b,*, Soung Hun Park a,c, Jesse C Craig a,b, Matthew T Lewis a,b, Joel D Trinity a,b,c, Russell S Richardson a,b,c, Ryan M Broxterman a,b,c
PMCID: PMC13560916  NIHMSID: NIHMS2205764  PMID: 40803624

Abstract

Skeletal muscle mitochondrial respiration is commonly assessed ex vivo using permeabilized fibers in media with high oxygen (O2) concentrations to ensure that O2 availability does not limit respiration. However, high O2 concentrations also increase the production of reactive O2 species that can negatively affect respiration. In this study, we tested the hypotheses that permeabilized fiber mitochondria in a high, compared to low, O2 concentration would (i) not be different at maximal state 3 respiration rate (Vmax), (ii) have lower submaximal respiration rates at submaximal O2 concentrations, and (iii) have greater total cumulative hydrogen peroxide (H2O2) appearance. We continuously monitored mitochondrial state 3 respiration and H2O2 appearance rates using high-resolution respirometry in permeabilized skeletal muscle fibers (12 untrained participants; 22 ± 4 yrs) with either control (~127 mmHg; CON) or high (~327 mmHg; HIGH) partial pressures of O2 (PO2). Vmax was not different between conditions (HIGH: 80.7 ± 16.7 vs. CON: 82.3 ± 18.7 pmol/s/mg, p = 0.695). The PO2 at 80 % Vmax (P80) was greater in HIGH (73.9 ± 25.5 vs. 28.0 ± 7.1 mmHg, p < 0.001) and respiration rates at 5–60 mmHg PO2 were lower for HIGH than CON (all p < 0.001). Additionally, the total cumulative H2O2 appearance was greater in HIGH than CON (n = 11; 51.5 ± 23.2 vs. 18.3 ± 10.3 pmol/mg, p < 0.001), and this difference was directly correlated with the difference in P80 (r = 0.655, p = 0.029). The current findings support that a high O2 concentration, by itself, does not appear to affect Vmax in the permeabilized skeletal muscle fiber preparation, but the corollary increase in H2O2 exposure may diminish mitochondrial state 3 respiratory function.

Keywords: Oxygen availability, Maximal respiration, Reactive oxygen species

1. Introduction

Mitochondria are critical organelles responsible for energy production in skeletal muscle through the generation of adenosine triphosphate (ATP) via aerobic respiration (i.e., mitochondrial respiration). Researchers routinely assess mitochondrial respiration using high-resolution respirometry to reveal the effects of diseases [1–3], interventions [4–6], and substrate utilization [7,8] on skeletal muscle mitochondrial bioenergetics. These studies commonly assess mitochondrial function in permeabilized muscle fibers ex vivo by measuring the rate of O2 consumption coupled to ATP production (i.e., state 3 respiration), which occurs in the presence of ADP and reflects active, phosphorylating respiration in response to energy demand [9–11]. State 3 respiration rate is dependent on O2 as a substrate for cytochrome c oxidase (complex IV), and experimental protocols typically utilize relatively high O2 concentrations in the respiration chamber to ensure O2 availability does not limit respiration [12]. Critically, these high O2 concentrations may directly or indirectly impair mitochondrial state 3 respiratory function, though this needs further investigation in permeabilized skeletal muscle fibers.

In skeletal muscle mitochondria, there is a complex relationship between the O2 concentration, production of reactive O2 species (ROS), and state 3 respiration rate. To elicit the maximal state 3 respiration rate (Vmax), the permeabilized skeletal muscle fiber technique requires relatively high O2 concentrations in the respiratory medium to avoid diffusion limitations caused by the distance between mitochondria embedded in the intact myofibrils and the respiration media [9,12]. A previous study found that O2 availability limited Vmax at a chamber partial pressure of O2 (PO2) of ~36 mmHg but not at ~140 mmHg in permeabilized human skeletal muscle fiber mitochondria [13]. That study also reported a greater mitochondria hydrogen peroxide (H2O2) appearance for the higher O2 concentration condition. Another study observed that mitochondrial H2O2 appearance was greater with higher O2 concentrations in permeabilized mouse skeletal muscle fibers [14]. Critically, the greater ROS production at high O2 concentrations may detrimentally affect mitochondria Vmax, submaximal state 3 respiration rate, or uncoupled respiration (i.e., LEAK respiration) [13,15,16], but this has not been elucidated in permeabilized human skeletal muscle fibers.

Therefore, this study investigated the effects of O2 availability on mitochondrial state 3 respiration and H2O2 appearance rates in permeabilized human skeletal muscle fibers. We specifically examined how control and high O2 concentrations affect mitochondrial state 3 respiration and H2O2 appearance rates across a dynamic range of O2 availability, starting at Vmax and continuing until respiration ceased. We tested the hypotheses that permeabilized fiber mitochondria in high, compared to control, would (i) not be different at Vmax, (ii) have lower submaximal respiration rates at submaximal O2 concentrations, and (iii) have greater total cumulative H2O2 appearance.

2. Methods

2.1. Ethical approval and participants

The Institutional Review Boards at the University of Utah and Salt Lake City Veterans Affairs Medical Center reviewed and approved this study, which conformed to the standards of the Declaration of Helsinki except for registration in a database. Participants were informed of the procedures and potential risks of participating in the study, and each participant provided verbal and written consent prior to participating in the study. A group of 12 participants (age: 22 ± 4 years; sex: 6 Female/6 Male; BMI: 24.7 ± 4.5 kg/m2) participated in the current study. All participants were free of overt chronic disease and did not engage in frequent exercise. The participants were instructed to fast for at least 4 h and refrain from vigorous exercise for at least 24 h prior to their experimental visit.

2.2. Skeletal muscle biopsy

Muscle samples were obtained under sterile conditions from the vastus lateralis, ~15 cm proximal to the knee. Approximately 10min after administering the local anesthetic (lidocaine), we used a sterile scalpel to make a small incision through which we inserted a 5-gauge biopsy needle to ~2cm below the muscle fascia. Immediately following the biopsy, 30–50 mg of the muscle was stored in ice-cold biopsy preservation fluid (BIOPS; 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 6.56 mM MgCl2, 0.5 mM DTT, 50 mM K-MES, 20 mM imidazole, 20 mM taurine, 5.77 mM Na2ATP, and 15 mM phosphocreatine, pH 7.1 at 4 °C) for the subsequent respiratory analyses.

2.3. Mitochondrial respiration and H2O2 production

Adipose and connective tissues were removed from the muscle fibers in 4 °C BIOPS by careful use of fine-tip forceps under a microscope (SZX10; Olympus, Center Valley, PA, USA). The muscle fibers were then permeabilized with Saponin in BIOPS (50 mg/ml) for 30 min. Saponin is a mild detergent that selectively permeabilizes the sarcolemma due to its high cholesterol content, while mitochondrial membranes remain unaffected at this concentration due to their lower cholesterol content [17]. Following permeabilization, the muscle fibers were rinsed twice for 10 min in mitochondrial respiration medium (MIR05; 2.77 mM CaK2EGTA, 7.23 mM K2EGTA, 6.56 mM MgCl2, 0.5 mM DTT, 20 mM imidazole, 5.77 mM ATP, 15 mM phosphocreatine, 50 mM K-MES, and 20 mM taurine, pH 7.0).

High-resolution respirometry (O2k, OROBOROS Instrument, Innsbruck, Austria) was used for the mitochondrial respiratory analyses, and all measurements were performed once per sample. Fig. 1 presents the experimental protocols and O2k tracings from a representative muscle sample. Briefly, permeabilized muscle fibers were placed in an O2k-chamber containing 2 ml of the MIR05 solution that was continuously stirred and maintained at 37 °C. For the control O2 concentration condition (CON), we left the respiration chamber open to room air until the O2 concentration in the MIR05 solution attained steady values (mean: 123 mmHg; range: 116–129 mmHg). For the high O2 concentration condition (HIGH), we injected 100 % O2 gas into the respiration chamber, before fully closing the stopper, to attain hyperoxic values in the MIR05 solution (mean: 327 mmHg; range: 295–425 mmHg). Respiration and H2O2 appearance rates were measured simultaneously throughout each experimental protocol. In the absence of ADP, mitochondrial Complex I + II LEAK respiration and H2O2 appearance rates were assessed following the addition of glutamate (2 mM), malate (10 mM), and succinate (10 mM). Next, ADP (2.5 mM) was added to the respiration chamber to assess Complex I + II state 3 respiration and H2O2 appearance rates. The protocol continued until respiration ceased, defined by a plateau in both O2 consumption and chamber O2 concentration, indicating that the mitochondria could no longer effectively utilize O2.

Fig. 1. Experimental protocols and Oxygraph-2 k tracings in permeabilized skeletal muscle fibers with control and high initial O2 concentrations in a representative sample.

Fig. 1.

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Panel a: CON, control initial PO2 (≈127 mmHg). Panel b: HIGH, high initial PO2 (≈327 mmHg). The blue lines represent the O2 concentration, while the red lines represent the O2 consumption in the respiration chamber. Substrates glutamate (2 mM), malate (10 mM), and succinate (10 mM) were added at the GMS indicator line to stimulate Complex I + II LEAK respiration. ADP (2.5 mM) was added at the ADP indicator line to stimulate Complex I + II state 3 respiration. The Vmax indicator line represents where Vmax was attained. The end indicator line represents where respiration ceased. Data shown are from one participant and are representative of the experimental protocols.

To measure the H2O2 and superoxide appearance rate from the mitochondria we added Amplex UltraRed (10 μM), horseradish peroxidase (1 U/ml), and superoxide dismutase (5 U/ml) to the respiration chamber prior to establishing to O2 concentration in the MIR05 solution. Catalyzed by the horseradish peroxidase, the Amplex UltraRed reacts with H2O2 in the MIR05 solution to form resorufin, which was measured using Fluorescence-Sensor Green of the LED2-Module (O2k-Fluo, OROBOROS Instrument, Innsbruck, Austria). The H2O2 data are presented as appearance, and not production, because the measurement only captures H2O2 in the solution and does not account for any ROS that is scavenged or remains in the mitochondria. Additionally, any superoxide in the respiration medium is converted to H2O2 by the added superoxide dismutase, so that the measured H2O2 values represented both H2O2 and superoxide appearance from the mitochondria. As H2O2 and superoxide are rapidly converted to resorufin, their concentrations in the respiration medium do not increase. Before each experiment, we generated a H2O2 standard curve to convert the fluorescence signal to a H2O2 appearance value.

2.4. Data and statistical analyses

Mitochondrial respiration and H2O2 appearance rates were corrected for non-mitochondrial background signals, which we measured after respiration ceased. These signals likely reflect non-specific fluorescence or residual chamber activity and therefore were subtracted from all preceding data. The rates were normalized to the dry muscle tissue mass, which was measured after the protocol ended. Mitochondrial respiration rate, chamber O2 concentration, and H2O2 appearance were measured every 2 s throughout each protocol from when respiration initially attained steady values until respiration ceased. We also averaged these data into bins for the range of chamber O2 concentration and the range of time that were common to both CON and HIGH. This range of chamber O2 concentrations was 0.1–60 mmHg, which was binned as 0.1–4.9, 5.0–10.0, 10.1–20.0, 20.1–30.0, 30.1–40.0, 40.1–50.0, 50.1–60.0 mmHg. The range of time was from 0 to 1100 s, which was separated into 100-s bins.

For both the CON and HIGH conditions, we determined the Vmax, PO2 corresponding to 80 % Vmax (P80), respiratory control rate (RCR), peak rate of H2O2 appearance, and cumulative H2O2 appearance. The RCR was calculated by state 3/LEAK respiration [18]. We determined Vmax as the highest respiration rate for each protocol. We determined the peak rate of H2O2 appearance as the highest rate for each protocol and the cumulative H2O2 appearance relative to time as the area under the curve above baseline for each protocol. Statistical analyses were performed using GraphPad Prism (Version 10.0.2; San Diego, CA). Two-way (bin x condition) repeated measures ANOVAs and Holm-Šídák post hoc testing were performed for both state 3 respiration rate and H2O2 appearance. Comparisons between CON and HIGH were made using paired samples t-tests for the Vmax, P80, RCR, peak rate of H2O2 appearance, and cumulative H2O2 appearance. We assessed the associations between select variables using Pearson correlations and we interpreted the strength of the correlation coefficients using previous recommendations [19]. For all statistical analyses, a p-value <0.05 was considered significant, and all data are expressed as mean ± standard deviation.

3. Results

3.1. Mitochondrial respiration in CON and HIGH

The individual data for mitochondrial respiration rates as a function of PO2 are presented in Fig. 2. While Vmax was not significantly different between CON and HIGH (p = 0.695, Figs. 3 & 4a), LEAK respiration was significantly greater in HIGH (27.6 ± 7.7 pmol/s/mg vs. 21.3 ± 5.1 pmol/s/mg, p < 0.001, Fig. 4a). RCR was significantly different between conditions (3.1 ± 0.5 HIGH vs 3.9 ± 0.8 CON, p < 0.001). There were significant effects for PO2 (p < 0.001), condition (p = 0.008), and their interaction (p < 0.001) for state 3 respiration rate. Specifically, the state 3 respiration rate was significantly lower in HIGH than CON for the 7.5–55 mmHg PO2 bins (p < 0.001, Fig. 3).

Fig. 2. Individual data documenting the effect of falling PO2 on mitochondrial respiration rate in permeabilized skeletal muscle fibers with control and high initial O2 concentrations.

Fig. 2.

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Panel a: CON, control initial PO2 (≈127 mmHg). Panel b: HIGH, high initial PO2 (≈327 mmHg). Corresponding colors between panels indicate the same participant.

Fig. 3. The effect of falling PO2 on mitochondrial respiration rate in permeabilized skeletal muscle fibers with control and high initial O2 concentrations.

Fig. 3.

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CON, control initial PO2 (≈127 mmHg); HIGH, high initial PO2 (≈327 mmHg). The symbols at the highest PO2 for each condition indicate the maximal mitochondrial state 3 respiration rates (Vmax). The dotted lines indicate the PO2 at 80 % Vmax (P80). Data are expressed as mean ± SD. n = 12 participants. * Significant difference between CON and HIGH at a given PO2, p < 0.05.

Fig. 4. The mitochondrial respiration rate, mitochondrial H2O2 appearance, and the ADP-induced decrease in H2O2.

Fig. 4.

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CON, control initial PO2 (≈127 mmHg); HIGH, high initial PO2 (≈327 mmHg). Panel a: Mitochondrial uncoupled respiration rate in the absence of ADP (LEAK) and maximal state 3 respiration rate (Vmax). Panel b: Mitochondrial H2O2 appearance at LEAK and Vmax. Panel c: H2O2 appearance normalized to respiration at LEAK and Vmax. Panel d: ADP-induced decrease in H2O2 appearance from LEAK to Vmax. Data are expressed as mean ± SD. * Significant difference between CON and HIGH, p < 0.05.

Additionally, the P80 was significantly greater in HIGH than CON (p < 0.001, Fig. 5a). The duration of the experimental protocol in HIGH was significantly longer than CON (43.4 ± 8.6 min vs. 21.3 ± 5.9 min, p < 0.001). Importantly, tissue weight was not significantly different between conditions (5.2 ± 1.1 mg in HIGH vs. 5.0 ± 1.1 mg in CON, p = 0.732).

Fig. 5. The PO2 at 80 % of the maximal mitochondrial respiration rate, and the total cumulative H2O2 appearance in permeabilized skeletal muscle fibers with control and high initial O2 concentrations.

Fig. 5.

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CON, control initial PO2 (≈127 mmHg); HIGH, high initial PO2 (≈327 mmHg). Panel a: PO2 at 80 % of Vmax (P80). Panel b: Total cumulative H2O2 appearance Panel c: The relationship between the absolute change between conditions for P80 and total cumulative H2O2 appearance. Data are expressed as mean ± SD. n = 12 participants for panel a; n = 11 participants for panels b & c.

3.2. Mitochondrial H2O2 appearance in CON and HIGH

All H2O2 data are for n = 11 because of technical issues during the protocols for one sample. The H2O2 appearance at both LEAK and Vmax was significantly greater for HIGH than CON (p < 0.001 and p = 0.007, respectively, Fig. 4b). When normalized to respiration rate, H2O2 appearance remained significantly greater in HIGH vs CON for at both LEAK and Vmax (p < 0.001 and p = 0.007, respectively, Fig. 4c). Additionally, the relative decrease in H2O2 appearance following the transition from LEAK to Vmax was significantly greater in HIGH compared to CON (p < 0.001, Fig. 4d).

There were significant effects for PO2 (p < 0.001), condition (p = 0.036), and their interaction (p < 0.001) for the rate of H2O2 appearance. Specifically, the rate of H2O2 appearance was significantly greater for CON than HIGH for the 35–55 mmHg PO2 bins (all p ≤ 0.028, Fig. 6a). There were also significant effects for time (p < 0.001), condition (p < 0.001), and their interaction (p < 0.001) for the cumulative H2O2 appearance relative to time. Specifically, these data revealed that the cumulative H2O2 appearance was significantly greater for HIGH than CON for the 350–1050 s time bins (all p < 0.011, Fig. 6b). The total cumulative H2O2 appearance was also significantly greater for HIGH than CON (p < 0.001, Figs. 5b & 6a). The absolute difference between conditions for the total cumulative H2O2 appearance was strongly correlated with the absolute change between conditions for the P80 (r = 0.693, p = 0.018, Fig. 5c).

Fig. 6. The effect of falling PO2 and time on mitochondrial hydrogen peroxide appearance in permeabilized skeletal muscle fibers with control and high initial O2 concentrations.

Fig. 6.

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CON, control initial PO2 (≈127 mmHg); HIGH, high initial PO2 (≈327 mmHg). Panel a: Mitochondrial hydrogen peroxide (H2O2) appearance at state 3 respiration across a falling PO2. Panel b: Mitochondrial cumulative H2O2 appearance at state 3 respiration over time. Data are expressed as mean ± SD. n = 11 participants. * Significant difference between CON and HIGH at a given PO2 or time, p < 0.05. † Significant difference between CON and HIGH for the peak rate of H2O2 appearance (Panel a) and the total cumulative H2O2 appearance (Panel b), p < 0.05.

4. Discussion

In the current study, we determined the impact of O2 availability on mitochondrial state 3 respiratory function in permeabilized human skeletal muscle fibers. To our knowledge, this is the first study to expose permeabilized muscle fiber mitochondria to CON (~123 mmHg) or HIGH (~327 mmHg) O2 concentrations and then measure state 3 respiration and H2O2 appearance rates until respiration ceased. In support of our first hypothesis, Vmax was not different between CON and HIGH. As Vmax was attained prior to a substantial increase in cumulative H2O2 appearance, no difference between conditions indicates that the higher O2 concentration by itself did not impair mitochondrial state 3 respiratory function. However, mitochondrial state 3 respiration rate was lower at submaximal PO2 values (7.55–55 mmHg) in HIGH compared to CON, supporting our second hypothesis. Together with the greater P80, these data indicate that mitochondrial state 3 respiration was impaired after attaining Vmax in HIGH. Further analyses revealed that LEAK respiration and LEAK H2O2 appearance were significantly greater, while RCR was significantly less, in HIGH compared to CON, which indicate decreased efficiency and increased ROS production linked to increased non-phosphorylating respiration. Additionally, the ratio between H2O2 appearance and respiration was greater in HIGH at both LEAK and state 3 respiration, further showing decreased efficiency in hyperoxia. Finally, the total cumulative H2O2 appearance was greater in HIGH than CON, consistent with our third hypothesis, and this was strongly related to the impairment in state 3 respiration in HIGH. In conclusion, a high O2 concentration, by itself, does not appear to affect Vmax in human permeabilized skeletal muscle fiber mitochondria, but the corollary increase in H2O2 exposure may diminish mitochondrial state 3 respiratory function.

4.1. Chamber O2 concentration and mitochondrial respiration

While many studies have explored the O2 dependence of mitochondrial respiration, most of this research was conducted using isolated mitochondria rather than permeabilized muscle fibers [20–25]. One advantage of isolated mitochondria is the negligible resistance to O2 diffusion, such that the generated oxygen kinetic curves accurately reflect cytochrome c oxidase [26]. However, an advantage of permeabilized fibers is that the in vivo cellular environment stays relatively intact [27,28]. Permeabilized muscle fibers are commonly utilized in studies that assess mitochondrial respiratory function via high-resolution respirometry [1,9,18]. Unlike isolated mitochondria, the permeabilized fibers have substantial resistance O2 diffusion between the respiration medium and the mitochondria [12,29]. To overcome this, studies routinely use high O2 concentrations (e.g., 177–355 mmHg) in the chamber to ensure that mitochondrial respiration is not constrained by O2 availability [9]. The fact that Vmax was not different between conditions in the current study (Figs. 3 & 4a) supports that O2 availability was not initially limiting in either condition. Although the initial O2 concentration in CON was sufficient to elicit Vmax, this was likely not high enough to avoid O2 availability limitations during a typical respiration protocol [13,14], confirming that higher O2 concentrations are likely necessary to maintain Vmax throughout these relatively long protocols.

4.2. Mitochondrial respiration and H2O2 exposure

The negligible resistance to the diffusion of O2 between the respiration medium and isolated mitochondria is such that the PO2 at cytochrome c oxidase is nearly equal to the PO2 in the respiration chamber [20,30]. This means that the hyperbolic relationship between mitochondrial respiration rate and PO2 can be accurately measured using chamber PO2, and researchers often characterize this curve using Michaelis-Menten kinetics or another regression model to indicate the PO2 at which respiration is limited by O2 availability (i.e., critical PO2) [31–33]. In permeabilized muscle fibers, however, the relatively greater resistance to O2 diffusion results in the PO2 at cytochrome c oxidase being less than the PO2 in the chamber and prevents the relationship between chamber PO2 and mitochondrial respiration rate from being hyperbolic (Fig. 2). Therefore, we opted to use a threshold PO2 value as an indication of an O2 availability limitation in mitochondrial respiration (P80) to avoid mathematical artifacts caused by the lack of Michaelis-Menten kinetics in permeabilized fibers. The P80 was 20 % greater in HIGH than CON (Fig. 5a). Interestingly, the P80 for HIGH does not appear to coincide with the point at which O2 availability limited respiration (Fig. 3). We base this on the assumption that the permeabilized muscle fiber O2 diffusion characteristics were similar for each condition and, therefore, a given PO2 in the chamber yielded similar PO2 values at cytochrome c oxidase. Thus, based on the P80 for CON, chamber PO2 values above 28 mmHg should have been enough for O2 availability not to limit Vmax in HIGH (Figs. 3 & 5a). The greater P80 together with the lower respiration rates across submaximal chamber PO2 values indicate that something other than O2 availability impaired state 3 respiration rate in HIGH. LEAK H2O2 production, state 3 H2O2 production, and cumulative H2O2 appearance were greater in HIGH than CON (Figs. 4b, 5b, & 6b). This is consistent with two important studies that found permeabilized muscle fiber mitochondria H2O2 appearance was directly related to chamber O2 concentration [13,14]. With greater chamber O2 concentrations, more O2 molecules are available in the mitochondria to interact with electrons at complexes I and III to form ROS [34,35]. Although mitochondria possess endogenous antioxidant systems, such as glutathione, peroxiredoxins, and superoxide dismutase, prolonged exposure to high O2 may overwhelm these buffering capacities, resulting in elevated H2O2 accumulation [36]. When ROS generation outpaces the capacity of these systems, oxidative stress ensues, potentially impairing mitochondrial function and cellular homeostasis. It is well documented that ROS production is closely linked to respiratory function in skeletal muscle mitochondria [37–39] and that ROS can damage key mitochondrial proteins involved in respiration [34,40,41]. High ROS concentrations can also promote mitochondrial uncoupling that reduces efficiency and impairs respiration if oxidative damage to mitochondrial components occurs [42,43]. Consistent with this, the difference in cumulative H2O2 appearance between HIGH and CON was directly related to the difference in P80 in the current study (Fig. 5d). Overall, these findings support that the total exposure of skeletal muscle mitochondria to H2O2 influences state 3 respiratory function.

5. Potential implications

The findings of the current study have important implications for studies using permeabilized muscle fibers to assess mitochondrial respiration. Many protocols (e.g., SUIT and ADP titration) vary in duration and require relatively high chamber O2 concentrations to ensure O2 availability does not limit mitochondrial respiration. The duration and O2 concentration will influence the total exposure of mitochondria to H2O2 throughout the protocol, which should be considered as part of data interpretation. For the protocols in the present study, Vmax was elicited early in the protocol before the mitochondria total H2O2 exposure substantially increased (Figs. 3 & 4a). Thus, the Vmax was not impaired in HIGH compared to CON, but the state 3 respiration rate was impaired later in the protocols after mitochondria total H2O2 exposure increased. This may not be the case for other protocols where Vmax occurs at a later time point. For these protocols, Vmax may be impaired, whereas the variables earlier in the protocol may not, because of the relation to mitochondria total H2O2 exposure. Interestingly, LEAK respiration and H2O2 appearance normalized to respiration were significantly greater in HIGH, which suggests that elevated O2 concentrations led to decreased efficiency and increased oxidative stress [11]. H2O2 appearance per unit of O2 consumption was also consistently greater during LEAK compared to Vmax in both conditions, with this difference being more pronounced in HIGH. Together, these findings suggest that hyperoxia not only increases proton conductance or electron leak, but also disproportionately enhances non-ATP-coupled H2O2 appearance, pointing to reduced coupling efficiency and elevated ROS production under high O2. The current data also indicate that the critical PO2 measured in permeabilized muscle fibers may be influenced by mitochondria total H2O2 exposure and not solely O2 availability. It is also important to consider that H2O2 appearance and ROS exposure will likely differ across participant cohorts and experimental conditions and, if not accounted for, may confound the mechanistic interpretation of differences in mitochondrial respiratory function. Thus, the exposure of mitochondria to ROS should be considered in studies assessing mitochondrial function ex vivo, particularly in studies where chamber O2 concentration varies throughout the protocol.

6. Experimental considerations

Several important factors should be considered when interpreting the results of this study. First, although we observed an association between elevated H2O2 exposure and impaired submaximal mitochondrial respiration, the absence of an antioxidant condition limits our ability to directly attribute this impairment to ROS. Second, the study design did not allow us to determine whether the impairment in respiration reflects a transient, reversible inhibition or irreversible mitochondrial damage. Addressing this question would require a recovery phase (i.e., reoxygenation) or sequential respiration protocols. Third, we assumed that diffusion characteristics of the samples were similar between CON and HIGH due to identical sample preparations, but prolonged exposure to elevated O2 and H2O2 concentrations in HIGH may have compromised mitochondrial membrane integrity as the protocol progressed. Fourth, we did not add cytochrome c to assess mitochondrial membrane integrity, as its addition at the start of the protocol could have interfered with subsequent respiration vs. PO2 measurements. Overall, the findings of this study provide compelling evidence that elevated H2O2 exposure can impair mitochondrial respiration and lay a strong foundation for future studies to determine the reversibility and mechanistic basis of this effect.

7. Summary and conclusion

This study is the first to comprehensively examine the impact of O2 concentration on mitochondrial state 3 respiration rate in permeabilized human skeletal muscle fibers, by exposing the mitochondrial to CON (~123 mmHg) or HIGH (~327 mmHg) O2 concentrations and then measuring respiration and H2O2 appearance rates until respiration ceased. In both conditions, Vmax was elicited early and was not different. After Vmax was attained in HIGH, mitochondrial state 3 respiration rates were impaired at submaximal PO2 values compared to CON. Mitochondrial total H2O2 exposure gradually increased throughout both protocols, and the total exposure was greater in HIGH than CON. The greater H2O2 exposure was directly related to the impairment in mitochondrial state 3 respiration in HIGH. In conclusion, a high O2 concentration, by itself, does not appear to affect Vmax in the permeabilized skeletal muscle fiber preparation, but the corollary increase in H2O2 exposure may diminish mitochondrial state 3 respiratory function.

Funding

This work was funded, in part, by the National Heart, Lung, and Blood Institute at the National Institute of Health (R01HL142603 and PO1 HL1091830), the Veterans Administration Clinical Science Research and Development Service (IK2CX002114 and I01CX00199), and Rehabilitation Research and Development Service (I02RX003810, IK2RX003913, E6910-R, E1697-R, E1433-P, E9275-L and E1572-P).

Footnotes

Declaration of competing interest

No conflict of interest.

CRediT authorship contribution statement

Bradley A. Ruple: Writing – review & editing, Writing – original draft, Visualization, Methodology, Investigation, Formal analysis, Data curation. Soung Hun Park: Writing – review & editing, Project administration, Methodology, Investigation, Data curation, Conceptualization. Jesse C. Craig: Writing – review & editing, Visualization, Validation, Project administration, Methodology, Data curation, Conceptualization. Matthew T. Lewis: Writing – review & editing, Project administration, Methodology, Data curation, Conceptualization. Joel D. Trinity: Writing – review & editing, Supervision, Investigation, Conceptualization. Russell S. Richardson: Writing – review & editing, Supervision, Resources, Methodology, Investigation, Conceptualization. Ryan M. Broxterman: Writing – review & editing, Supervision, Project administration, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Data availability

Data will be made available on request.

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