Mitochondrial free radicals contribute to cigarette smoke condensate-induced impairment of oxidative phosphorylation in the skeletal muscle in situ

Abstract

Oxidative stress plays a critical role in cellular dysfunction associated with cigarette smoke exposure and aging. Some chemicals from tobacco smoke have the potential to amplify mitochondrial ROS (mROS) production, which, in turn, may impair mitochondrial respiratory function. Accordingly, the present study tested the hypothesis that a mitochondria-targeted antioxidant (MitoTEMPO, MT) would attenuate the inhibitory effects of cigarette smoke on skeletal muscle respiratory capacity of middle-aged mice. Specifically, mitochondrial oxidative phosphorylation was assessed using high-resolution respirometry in permeabilized fibers from the fast-twitch gastrocnemius muscle of middle-aged C57Bl/6J mice. Before the assessment of respiration, tissues were incubated for 1hr with a control buffer (CON), cigarette smoke condensate (2 % dilution, SMOKE), or MitoTEMPO (10 μM) combined with cigarette smoke condensate (MT + SMOKE). Cigarette smoke condensate (CSC) decreased maximal-ADP stimulated respiration (CON: $60\pm 15{\text{pmolO}}_2\cdot {\text{s}}^{-1}\cdot {\text{mg}}^{-1}$ and $\text{SMOKE:}33\pm 8{\text{pmolO}}_2{\text{s}}^{-1}\cdot {\text{mg}}^{-1};\text{p}=0.0001$), and this effect was attenuated by $\text{MT}$ $(\text{MT}+\text{SMOKE:}41\pm 7{\text{pmolO}}_2\cdot {\text{s}}^{-1}\cdot {\text{mg}}^{-1};\text{p}=0.02\text{with SMOKE})$. Complex-I specific respiration was inhibited by CSC, with no significant effect of MT (p = 0.35) Unlike CON, the addition of glutamate (ΔGlutamate) had an additive effect on respiration in fibers exposed to $\text{CSC}\left(\text{CON}:0.9\pm 1.1{\text{pmolO}}_2\cdot {\text{s}}^{-1}\cdot {\text{mg}}^{-1}\text{and SMOKE}:5.4\pm 3.7{\text{pmolO}}_2\cdot {\text{s}}^{-1}\cdot {\text{mg}}^{-1};\text{p}=0.008\right)$ and $\text{MT}(\text{MT}+\text{SMOKE}:8.2\pm 3.8{\text{pmolO}}_2\cdot {\text{s}}^{-1}\cdot {\text{mg}}^{-1};\text{p}\le 0.01)$. Complex-II specific respiration was inhibited by CSC but was partially restored by MT (p = 0.04 with SMOKE). Maximal uncoupled respiration induced by FCCP was inhibited by CSC, with no significant effect of MT. These findings underscore that mROS contributes to cigarette smoke condensate-induced inhibition of mitochondrial respiration in fast-twitch gastrocnemius muscle fibers of middle-aged mice thus providing a potential target for therapeutic treatment of smoke-related diseases. In addition, this study revealed that CSC largely impaired muscle respiratory capacity by decreasing metabolic flux through mitochondrial pyruvate transporter (MPC) and/or the enzymes upstream of α-ketoglutarate in the Krebs cycle.

1. Introduction

Cigarette smoke (CS) exposure is still estimated to cause more than 480,000 deaths annually in the United States and is a primary risk factor for several cancers, respiratory, cardiovascular, and chronic metabolic diseases [1]. Epidemiological studies have consistently demonstrated a strong association between CS and the risk for metabolic diseases in middle-aged and older adults [1]. Knowledge of the biological mechanisms underlying the pathogenic effects of cigarette smoke on health therefore continues to be of great importance to reduce disease burden from CS exposure. In this context, it is noteworthy that the extra-pulmonary manifestations of cigarette smoke exposure, which significantly contribute to poor health outcomes and increased mortality in smokers, are still poorly understood.

Cigarette smoke (CS) is composed of approximately 7357 different chemicals [2], including reactive oxygen species (ROS) and numerous toxicants. Some of these compounds can cross the alveolar-capillary barrier in the lungs, diffuse into the bloodstream, and reach other organs and peripheral tissues in the body causing toxicity [3]. Because mitochondria are present in almost every cell of the body and are essential to maintain cellular homeostasis, they are implicated as both a target and a mediator of cigarette smoking’s deleterious impact on cardio-metabolic health [4,5]. Although mitochondria endogenous antioxidant system is extensive (e.g. manganese superoxide dismutase, glutathione, thioredoxin, catalase), elevated ROS levels associated with chronic exposure to cigarette smoke can overwhelm its capacity to scavenge free radicals resulting in oxidative damage and lipid peroxidation [6,7]. Specifically, besides the direct effects of some chemicals contained by cigarette smoke (e.g. cyanide, nicotine, o-cresol, decanoic acid) on mitochondrial oxidative phosphorylation [8], CS may enhance mitochondrial-derived ROS production, thus triggering a vicious cycle that promotes oxidative stress and aggravates bioenergetic deficits [9, 10]. This effect might also be amplified with advancing age due to the greater susceptibility of mitochondria to redox stress [11,12].

However, testing this hypothesis requires simultaneously measuring mitochondrial respiratory rate and free radical production in the physiological environment of the tissues in situ. Detection of free radicals during respiration is technically challenging due to their low concentration under physiological conditions and high reactivity with other molecules. The detection of extracellular hydrogen peroxide (H₂O₂) production with amplex red using fluorescence is an attractive approach to assess the redox state in permeabilized tissues [13]. This technique is, however, hampered by the interaction of mitochondrial H₂O₂ with O₂ concentration and the activity of endogenous antioxidant enzymes in the mitochondrial matrix [14], which complicates the accurate quantification of mitochondrial-derived ROS. Alternatively, electron paramagnetic resonance (EPR) spectroscopy is both sensitive and specific to detect mitochondrial free radicals [15]. However, this method is better suited for steady-state conditions as this technique requires immediate freezing of tissue samples. In this context, mitochondrial-targeted antioxidants represent an interesting experimental approach to examining the contribution of mitochondrial ROS in mediating the metabolic effects of cigarette smoke.

Accordingly, this study aimed to determine the effects of a mitochondria-targeted antioxidant (MitoTEMPO) on mitochondrial oxidative phosphorylation in permeabilized skeletal muscle fibers acutely exposed to cigarette smoke in middle-aged mice. Based on prior studies [8,16,16–19], we hypothesized that CSC would inhibit mitochondrial respiration. However, MitoTEMPO would attenuate the inhibitory effect of CSC on maximal ADP-stimulated respiration rate by increasing the highly redox-sensitive complex-I linked respiration [20].

2. Methods

2.1. Animals and experimental design

Middle-aged (10.9 ± 0.8 months) C57BL/6J mice (n = 16, 9 male/7 female) were used for this study [21]. The age of these mice was selected based on epidemiological evidence demonstrating that the prevalence for smoking is highest in middle-aged adults [22], and concomitant to a sharp rise in COPD prevalence [22,23]. This period is therefore critical to identify key cellular targets for interventions that can alter the long-term trajectory for adults at risk of developing lung disease. All animals were maintained on a 12-h dark/light cycle without access to running wheels and were fed standard chow ad libidum. After euthanasia by 5 % isoflurane, the white gastrocnemius was subsequently harvested and immediately placed in an ice-cold BIOPS preservation solution [24]. The white gastrocnemius was chosen due to its middling susceptibility to cigarette-smoke induced metabolic disruption [16], which makes this muscle both a sensitive and generalizable model for evaluating the interaction between CS toxicity and mitochondrial-derived ROS. Protocols were approved by the Institutional Animal Care and Use Committee of UMASS Amherst.

2.2. Chemical preparations

Cigarette Smoke Condensate (CSC; Murty Pharmaceuticals, Lexington, KY) containing 40 mg of cigarette smoke particulate matter per milliliter was diluted in MiR05 (in mM: 110 Sucrose, 0.5 EGTA, 3 MgCl2, 60 K-lactobionate, 20 taurine, 10KH2PO4, 20 HEPES, BSA 1 g L⁻¹, pH 7.1) to a concentration of 800 μg/mL (2 % CSC). MitoTEMPO (MT) is composed of piperidine nitroxide, a compound with antioxidative properties, and triphenylphosphonium, which allows the crossing of cellular membranes and the mitochondrial localization of the drug [25] that act as a superoxide dismutase mimetic to scavenge free radicals in the mitochondria. MitoTEMPO (MT) powder was purchased from Sigma Aldrich (CAS no. 1334850–99-5) and prepared before each experiment for co-incubation with CSC (10 μM MT + CSC).

2.3. Preparation of permeabilized muscle fibers

The tissue preparation and respiration measurement techniques were adapted from established methods [24,26] and have previously been described in detail by our group [16,27,28]. After brief immersion in BIOPS (in mM: 2.77 CaK2EGTA, 7.23 K2EGTA, 50 K + MES, 6.56 MgCl2, 20 Taurine, 5.77 ATP, 15 mM PCr, 0.5 DTT, 20 Imidazole), muscle fibers were carefully separated with fine-tip forceps, and then immersed in a BIOPS-based saponin solution (50 μg saponin⋅mL⁻¹ BIOPS) for 30 min at 4 °C. Following saponin permeabilization, fiber bundles were then bathed twice in ice-cold mitochondrial respiration fluid (MiR05 in mM: 110 Sucrose, 0.5 EGTA, 3 MgCl2, 60 K-lactobionate, 20 taurine, 10KH2PO4, 20 HEPES, BSA 1 g L⁻¹, pH 7.1) for 10 min each.

After chemical permeabilization, fiber bundles were incubated for 1 h in a 2 mL solution of MiR05 (control), MiR05 with 2 % (800 μg/mL) cigarette smoke concentrate (CSC; Murty Pharmaceuticals, Lexington, KY), or MiR05 with 2 % CSC and 10 μM MitoTEMPO (MT + CSC; Sigma Aldrich) at 4 °C. The concentration of MitoTEMPO was based on its known IC₅₀ (10 μM) for mitochondrial superoxide [29] and previous studies demonstrating a physiological effect of MitoTEMPO on vascular function at even higher concentration (1 mM) [30,31]. A set of pilot experiments confirmed the effectiveness of 10 μM in our in situ preparation.

Following the treatment incubation, fiber bundles were gently dabbed with a paper towel to remove excess liquid, the wet weight of the sample (1–2 mg) was then measured using a standard calibrated scale. Fiber bundles were placed in the respiration chamber (Oxygraph O2K, Oroboros Instruments, Innsbruk, Austria) with 2 mL of MIR05 solution warmed to 37 °C. Pure oxygen gas was then added to both chambers to maintain an oxygen concentration of 190–300 μM during the protocol to prevent any O₂ diffusion limitation. Once the permeabilized fiber bundles reached a state of equilibrium after ~5–10 min, the mitochondrial respiratory function was assessed in duplicate. After the titration of each substrate, the steady-state respiration rate was averaged for at least 30 s. The rate of O₂ consumption was normalized to the wet weight of the tissue [picomoles per second per milligram of wet weight (JO₂ in pmolO₂/sec/mg_wt)].

2.4. Respiration protocol

The titration protocol was designed to comprehensively investigate the components of oxidative phosphorylation in permeabilized muscle fibers and was performed as follows: saturating concentrations of pyruvate (P; 5 mM) and malate (M; 2 mM) were first added to the chambers to assess non-phosphorylating oxygen consumption. After a steady state was reached, saturating amounts of adenosine diphosphate (ADP; 5mmM) were added to activate ATP synthase. Then, glutamate (G; 10 mM), which provides electrons to complex I from the metabolism of oxaloacetate in the citric acid cycle (TCA) was added. ΔGlutamate was calculated by subtracting the respiration rates of PMDG from the respiration rates of PMD (PMDG – PMD) to determine the additive effect of glutamate on respiration. This step was followed by the addition of succinate (S; 10 mM) a complex II agonist. At this point in the protocol, maximal ADP-stimulated respiration with convergent electron flow through complex I and II was measured. Cytochrome c (C; 10 μM) was subsequently added to test the mitochondrial membrane integrity [24, 26]. Any samples that demonstrated an increase in respiration above 10 % after the addition of cytochrome c were excluded from the analysis. Next, carboxyatractyloside (CAT; 5 μM, Cayman Chemical no. 21120), a selective non-competitive inhibitor of the adenine nucleotide translocase (ANT), was added. The role of ANT on respiration was determined by subtracting maximal ADP-stimulated respiration from respiration after the ANT inhibitor carboxyatractyloside (CAT) was added, divided by peak ADP stimulated respiration times 100 % [(PMDGS-CAT/PMDGS) *100 %]. Carbonyl cyanide m-chlorophenyl hydrazone (FCCP) was titrated to assess the uncoupled capacity of the mitochondrial electron transport chain. Rotenone (Rot; 0.5 μM), a complex-I inhibitor was titrated to assess complex-II driven respiration. The relative contribution of complex II to overall uncoupled respiration was calculated as the respiration rates after the addition of ROT (CI Inhibitor) divided by FCCP-induced uncoupled respiration times 100 % [(ROT/FCCP)*100 %]. Finally, oligomycin (Omy; 2.5 μM), an inhibitor of ATP synthase, and antimycin A (AmA; 2.5 μM), a complex III inhibitor, were added to the chambers to assess residual, or non-mitochondrial, oxygen consumption.

2.5. Data analysis

Outliers were first assessed using a ROUT test (Q = 10 %) and excluded from analysis if necessary. Normality was tested using a Shapiro-Wilk test. The effects of treatment (CSC/MT + CSC) on mitochondrial respiration were determined using a repeated one-way ANOVA with the Geisser-Greenhouse correction, followed by a post hoc Holm-Šidák adjustment. If extreme outliers were identified and excluded from analysis leaving a missing value, a mixed effect model analysis was used followed by a post hoc Holm-Šidák adjustment. Results are presented as mean ± standard deviation (SD) in the text. Statistical significance was determined as *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. Mean values are shown as bars with individual data points to illustrate the relative distribution of data points in the figures. Prism 10.0 – GraphPad was used to perform statistical analysis and create respective figures.

3. Results

Collective respiration rates (or percent change) are summarized in Table 1.

Table 1.

Summary of the effects of cigarette smoke condensate (CSC) and MitoTEMPO on mitochondrial respiration

		Respiration rate (JO2) in pmolO2/sec/mgwt
Substrate	Respiratory Complex/State	Control (CON)	Cigarette Smoke Condensate (CSC)	MitoTEMPO + CSC (MT + CSC)
PM	Leak Respiration	14.4 ± 2.8	12.2 ± 4.9	14.2 ± 4.2
PMD	Complex I	46.0 ± 13.7	20.2 ± 5.2****	22.1 ± 3.3****
PMDG	Complex I	50.1 ± 16.3	25.6 ± 6.1***	30.7 ± 5.5**,†
ΔGlutamate		0.9 ± 1.1	5.4 ± 3.7**	8.2 ± 3.8****,†
PMDGS + ROT	Complex II	38.8 ± 9.5	22.2 ± 5.1****	26.9 ± 6.8**,†
PMDGS	State II	60.1 ± 15.1	33.9 ± 8.4****	40.7 ± 6.7**,†
FCCP	Uncoupled	70.6 ± 11.8	38.9 ± 13.1****	44.6 ± 13.1****
	CII Percent Contribution to Uncoupled	55 ± 8 %	60 ± 10 %	60 ± 8 %
	CAT Inhibition Percent of State III	57 ± 11 %	20 ± 17 %****	20.93 ± 7.88 %****

Note. This table summarizes the main effects of cigarette smoke condensate (CSC) and the mitochondria-targeted antioxidant MitoTEMPO on various steps of mitochondrial respiration in permeabilized skeletal muscle fibers from middle-aged mice. The parameters assessed include leak respiration, ADP-stimulated respiration at complex I and II specific states, and uncoupled respiration as the mean ± standard deviation in picomoles per second per milligram of wet weight (JO₂ in pmolO₂/sec/mg_wt). Complex II contribution to total uncoupled respiration and CAT inhibition of State III is also shown as a percentage. Statistical significance indicated as

^*p ≤ 0.05

^**p ≤ 0.01

^***p ≤ 0.001

^****p ≤ 0.0001 with CON, and

^†p ≤ 0.05

^††p ≤ 0.01

^†††p ≤ 0.001

^††††p ≤ 0.0001 with CSC.”

3.1. Effects of CSC and MitoTEMPO on leak respiration

Non phosphorylating leak respiration rates (JO₂) with pyruvate and malate (PM_LEAK) were not statistically different between any of the conditions (CONTROL (CON): 14.4 ± 2.8 pmol s⁻¹.mg_wt⁻¹; CSC: 12.2 ± 4.9 pmol s⁻¹.mg_wt⁻¹; MT + CSC: 14.16 ± 4.2 pmol s⁻¹.mg_wt⁻¹; p = 0.20).

3.2. Effects of CSC and MitoTEMPO on maximal ADP-stimulated respiration

Respiration rates (JO₂) for maximal ADP-stimulated respiration (PMDGS) are illustrated in Fig. 1. Results showed a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC significantly inhibited maximal ADP-stimulated respiration compared with control $(\text{CON}:60.1\pm 15.1{\text{pmol s}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and}\text{CSC}:33.9\pm 8.4{\text{pmols}}^{-1}.{\text{mg}}_{\text{wt}}^{-1};\text{p}=0.001)$. This effect was attenuated by $\text{MitoTEMPO}(\text{MT}+\text{CSC}:40.7\pm 6.7{\text{pmols}}^{-1}.{\text{mg}}_{\text{wt}}^{-1};\text{p}=0.02\text{with CSC})$ which increased respiration compared with CSC. Fibers treated with MitoTEMPO still demonstrated a significantly lower respiration rate when compared with the control fibers (p = 0.001 with CONTROL).

Fig. 1.

Maximal ADP-stimulated mitochondrial respiration in CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 14–16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown.

3.3. Effects of CSC and MitoTEMPO on glutamate metabolism

Fig. 2A displays respiration rates (JO₂) with pyruvate (P), malate (M), and ADP (D). There was a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC significantly inhibited mitochondrial respiration linked to PMD $\left(\text{CON}:46.0\pm 13.7{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and CSC}:20.2\pm 5.2{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=<0.0001\right)$, but MitoTEMPO was not significantly different from $\text{CSC}(\text{MT}+\text{CSC}:22.1\pm 3.3{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.35\text{with CSC})$. Fig. 2B displays respiration rates (JO₂) with pyruvate (P), malate (M) ADP (D), and glutamate (G). There was a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC significantly inhibited mitochondrial respiration supported by $\text{PMDG}\left(\text{CON}:50.1\pm 16.3\text{pmol}{\text{s}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and CSC}:25.6\pm 6.1{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.002\right)$, this effect was attenuated by $\text{MitoTEMPO}(\text{MT}+\text{CSC}:30.7\pm 5.5{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.03\text{with CSC})$which increased respiration rates compared with CSC. Fig. 2C depicts the additive effect of glutamate (G) on respiration. Analysis indicated that there was a significant main effect of treatment on respiration (p <0.0001). Post hoc analysis indicated that the addition of glutamate (ΔGlutamate, 10 mM) to pyruvate (P), malate (M) and ADP (D) had no significant effect on control fibers $\left(\text{CON}:0.92\pm 1.08\text{pmol}{\text{s}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\right)$. In contrast, glutamate exerted an additive effect on respiration in fibers exposed to $\text{CSC}\left(\text{CON}:0.9\pm 1.1{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and SMOKE}:5.4\pm 3.7{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.008\right)$ and $\text{MT}\left(\text{MT}+\text{SMOKE}:8.2\pm 3.8{\text{pmols}}^{-1}.{\text{mg}}_{\text{wt}}^{-1};\text{p}=0.01\text{with SMOKE},\text{p}<0.0001\text{with CON}\right)$.

Fig. 2.

Respiration stimulated by pyruvate, malate, ADP (A), and glutamate (B). ΔGlutamate was calculated as PMDG-PMD (C). CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 13–16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown.

3.4. Effects of CSC and MitoTEMPO on Complex-II and uncoupled respiration

The respiration rates for complex-II linked respiration are displayed in Fig. 3. Results from the repeated one-way ANOVA showed a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC had an inhibitory on skeletal muscle respiration rates $\left(\text{CON}:38.8\pm 9.5{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and SMOKE}:22.2\pm 5.1{\text{pmol s}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}<0.001\right)$. This effect was attenuated by $\text{MitoTEM}\left(\text{MT}+\text{CSC}:26.9\pm 6.8{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.03\text{with SMOKE}\right)$. Uncoupled respiration rates induced by FCCP are shown in Fig. 4. Analysis indicated that there was a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC inhibited uncoupled respiration $\left(\text{CONTROL}:70.6\pm 11.8{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1}\text{and SMOKE}:38.9\pm 13.1{\text{pmols}}^{-1}.{\text{mg}}_{\text{wt}}^{-1};\text{p}<0.001\right)$, and MitoTEMPO did not significantly alter this inhibitory effect $\left(\text{MT}+\text{CSC}:44.6\pm 13.1{\text{pmols}}^{-1}\cdot {\text{mg}}_{\text{wt}}^{-1};\text{p}=0.17\text{with SMOKE}\right)$. The relative contribution of complex II to overall uncoupled respiration is shown in Fig. 5. Results were not statistically different between treatment conditions (CON: 55 ± 8 %; CSC: 60 ± 10 %; MT + CSC: 60 ± 8 %; p = 0.12).

Fig. 3.

Complex-II driven respiration CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown.

Fig. 4.

FCCP induced uncoupled respiration. CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown.

Fig. 5.

Relative contribution of complex-II to peak uncoupled respiration. CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 15–16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown.

3.5. Effects of CSC and MitoTEMPO on the adenosine nucleotide translocator (ANT)

The effects of treatment (CSC/MT + CSC) on the mitochondrial ANT are displayed in Fig. 6. There was a significant main effect of treatment on respiration (p < 0.0001). Post hoc analysis indicated that CSC had a significant inhibitory effect on ANT-mediated respiration (CON: 57 ± 11 % and SMOKE: 20 ± 17 %; p < 0.0001). MitoTEMPO did not significantly affect the ANT-mediated respiration (MT + CSC: 20.93 ± 7.88 %; p = 0.83 with SMOKE).

Fig. 6.

Percent CAT inhibition calculated as ((PMDGS-CAT)/PMDGS)*100 %. CON (white/●), CSC (dark gray/∎), and MT + CSC (light gray/▴) from the gastrocnemius muscle (n = 14–16 per group) expressed as mean. Significant or trending p-values from post hoc analysis are shown. CAT, carboxyatractyloside.

4. Discussion

In the present study, we sought to determine the role of mitochondrial ROS in mediating the metabolic effects of cigarette smoke in middle-aged mice by using a mitochondria-targeted antioxidant in permeabilized skeletal muscle fibers acutely exposed to cigarette smoke condensate. As previously documented, acute cigarette smoke concentrate exposure significantly inhibited ADP-stimulated respiration supported by complex I in the fast-twitch gastrocnemius muscle [16–19]. However, unique to the middle-aged mice was the finding that complex II-linked respiration was also impaired following cigarette smoke concentrate exposure. MitoTEMPO, a mitochondrial-targeted antioxidant, partially attenuated the effects of CSC on maximal ADP-stimulated respiration. This protective effect of MitoTEMPO was mediated by an independent increase in complex I- and complex II- supported respiration, whereas the ANT-mediated exchange of ATP/ADP across the inner mitochondrial membrane remained impaired by CSC. Interestingly, the addition of glutamate exerted an additive effect on respiration in fibers exposed to CSC and MitoTEMPO thus suggesting the stimulation of enzymes downstream of α-ketoglutarate in the TCA cycles. Together, these findings suggest that mitochondrial ROS (mROS) significantly contributed to cigarette smoke-induced inhibition of respiration. However, it also hints at the existence of additional mechanisms responsible for smoke-induced metabolic impairment in the mitochondria, as increasing the mitochondria’s ability to scavenge free radicals did not fully restore muscle respiratory capacity in middle-aged mice.

4.1. Cigarette smoke condensate impaired skeletal muscle respiratory capacity in middle-aged mice

4.1.1. CSC inhibition of maximal ADP-stimulated respiration and complex I respiration

Consistent with our hypothesis, cigarette smoke condensate (CSC) substantially impaired ADP-stimulated respiration capacity using various combination of substrates in permeabilized fibers from the fast-twitch gastrocnemius muscle. Specifically, CSC inhibited convergent electron flow through complex I and II by ~55 % (PMDGS, Fig. 1) and complex I-linked respiration by ~44 % (pyruvate only, Fig. 2A). Consistent with these results, the capacity for electron transfer through the respiratory complexes was also substantially impaired as uncoupled respiration with the protonophore FCCP was ~41 % lower with CSC (Fig. 4). These results are in agreement with previous work using acute smoke exposure in permeabilized fibers from the gastrocnemius muscle, which documented that CSC inhibited ADP stimulated respiration by ~58 % and 40 % with pyruvate or glutamate substrates to support electron flux through complex-I, respectively [16,17,17–19].

It is noteworthy that in the present study CSC concentration was reduced by half (2 % versus 4 % previously), and yet yielded similar impairment in muscle respiratory capacity compared with previous studies [16,17,17–19]. Given the previously documented dose-dependent effect of CSC at this concentration in the gastrocnemius [16–19], and the similar in situ preparation used, these results suggest a greater susceptibility of the skeletal muscle of middle-age mice to the inhibitory effect of cigarette smoke. In support of this interpretation, previous studies have documented that aging was associated with greater mitochondrial susceptibility to dysfunction and structural anomalies following xenobiotic exposure such as particulate matter 2.5 or vanadium [32,33]. Together, these findings highlight the role of aging in increasing the vulnerability of mitochondria to cellular stressors, which in turn can exacerbate the adverse cellular outcomes, including impaired energy homeostasis, related to xenobiotic exposure.

4.1.2. Glutamate-specific stimulation of mitochondrial respiration with CSC

A unique aspect of the titration protocol in the present study was the separate assessment of pyruvate and glutamate-driven respiration. Both substrates activates dehydrogenases resulting in the reduction of NADH to NAD + for the transfer of electron through complex I [34]. However, pyruvate and glutamate operate as metabolic intermediates at different steps of the citric acid cycle (TCA) and thus can provide specific information on the rate-limiting steps. Specifically, pyruvate import is mediated through the outer mitochondrial membrane via the voltage-dependent anion channel (VDAC), then through the inner mitochondrial membrane by the mitochondrial pyruvate carrier (MPC) for oxidation to acetyl CoA within the matrix via the pyruvate dehydrogenase complex, i.e. the first step of the TCA cycle [35]. Alternatively, glutamate undergo conversion into α-ketoglutarate, an intermediate involved in downstream biochemical reactions of the TCA cycle [36].

In the control condition, glutamate did not significantly alter ADP-stimulated respiration already supported by pyruvate and malate oxidation. However, after CSC incubation, titration of glutamate elicited an additive effect, with NADH-linked respiration increasing by as much as 28 % (Fig. 2C). This finding points out to some rate-limiting steps upstream of α-ketoglutarate implicating the pyruvate carrier and/or other enzymes that precede α-ketoglutarate in the TCA cycle. In this line, nicotine and acrolein, two toxic compounds found in cigarette [8,37] have been documented to inhibit pyruvate dehydrogenase activity in mitochondria from pancreatic or liver tissues [37,38]. In addition, the enzymatic activity of aconitase, a redox-sensitive enzyme involved in the mitochondrial TCA cycle that regulates mitochondrial metabolism [39], was decreased by nicotine exposure in pancreatic cells [25]. Based on these studies and the present results, mitochondrial pyruvate transporter (MPC) and/or the enzymes upstream of α-ketoglutarate in the Krebs cycle appears to be a primary target of cigarette smoke toxicity.

4.1.3. CSC inhibition of complex II-linked respiration

Another novel finding from this study was that CSC inhibited Complex II linked respiration by ~56 % (Fig. 3). This result is somewhat at odd with previous work from our group [16,17,17–19] and others [8]. In these studies, complex II supported respiration in the skeletal muscle remained mostly unaltered by acute cigarette smoke exposure. This discrepancy can, however, be explained by the different age of the mice between the studies (mature adult versus middle-aged), which lend further credence to the hypothesis that age-related alteration in mitochondrial function decrease the ability of the skeletal muscle to cope with transient oxidative stress and xenobiotic exposure such as cigarette smoke. In support of this view, low dose of paraquat, a chemical agent used to induce a mild acute oxidative insult, decreased ATP levels and resulted in greater metabolic disturbance in the skeletal muscle of middle-aged mice compared with younger mice [12]. Of note, CSC had a larger effect on complex I- than complex II- linked respiration in the present study suggesting that complex I is the most vulnerable site to xenobiotic and/or oxidative stressors [40]. Together, these findings suggest that the greater vulnerability of the mitochondria to stress with advancing age leads to the development of energetic deficits, which may explain some of the skeletal muscle derangements and greater fatigability associated with chronic smoking in middle-age and older adults [41,42].

4.2. MitoTEMPO attenuated CSC-induced inhibition of TCA flux but not mitochondrial phosphate transport

The mitochondria have been reported to be the main producer of endogenous ROS within the cell, responsible for around 90 % of all ROS production [43]. An impaired mitochondrial function may lead to excess ROS production at the mitochondria, especially through complexes I and III [44]. In the present study, treatment with the mitochondrial-targeted antioxidant MitoTEMPO partially mitigated the effects of acute CSC exposure on mitochondrial respiration in the gastrocnemius muscle. Specifically, maximal ADP-stimulated respiration with convergent electron flow through complex I and II was ~25 % higher in MT + CSC than CSC (Fig. 1). Interestingly, complex I-specific respiration supported by pyruvate/malate was not significantly different between MT + CSC and CSC (Fig. 2A). However, when glutamate was titrated, MitoTEMPO partially restored complex I driven respiration compared with controls and CSC conditions (Fig. 2B and C). In addition, complex II linked respiration with succinate was also significantly higher (~30 %) in MT + CSC compared with CSC (Fig. 3).

These findings suggest that MitoTEMPO exerts some protective effects on several enzymes downstream of α-ketoglutarate in the TCA cycles and/or the electron transport chain. Given that uncoupled respiration with FCCP in CSC and MT + CSC conditions were significantly lower than control (Fig. 4), but their rates were still superior to those of maximal ADP-stimulated respiration (Fig. 1), it is unlikely that the electron transport system capacity was the main rate-limiting step affected by MitoTEMPO. Alternatively, the improvement in glutamate- and succinate-linked respiration supports the interpretation that the enzymes downstream of α-ketoglutarate in the TCA cycle were most likely sensitive to the antioxidant effects of MitoTEMPO. Future studies focused on enzymatic activity and metabolomics are therefore warranted to identify the exact steps of the TCA cycle most affected by cigarette smoke exposure and the associated changes in redox balance.

Recently, adenosine nucleotide translocase (ANT), which is responsible for the ADP/ATP exchange across the mitochondrial membrane and mitochondrial coupling (Bertholet et al., 2019), has been identified as a primary target of cigarette smoke toxicity [16–19]. In the present study, we used carboxyatractyloside (CAT) to evaluate ANT-mediated respiration linked to exchange of ADP/ATP across the mitochondrial membrane. In the control condition, CAT inhibited respiration by ~60 % (Fig. 6). This effect was lessened by CSC exposure (~20 %), even with co-incubation with MitoTEMPO. The lack of effect of MitoTEMPO therefore rules out any redox-dependent mechanism to explain the inhibitory effect of CSC on ANT function and the exchange of ADP/ATP across the membrane and rather points out to a direct inhibition of ANT by CSC. Given the key role played by ANT in controlling oxidative phosphorylation rate, these results also explain the small magnitude of the effects of MitoTEMPO on maximal ADP-stimulated respiration (~25–30 %) as the inhibition of ANT function may become the predominant rate-limiting step for the production of ATP when glutamate- and succinate-linked respiration are partially restored by MitoTEMPO.

Overall, these results imply that mitochondria, especially the TCA cycle enzymes and ADP/ATP exchange mechanism across the mitochondrial membrane, are sensitive allosteric targets to cigarette smoke condensate toxicity. However, our findings with MitoTEMPO demonstrate that mitochondria also contribute to tissue and organ dysfunction through enhanced mitochondrial ROS production that amplifies the bioenergetic deficits caused directly by cigarette smoke. Consistent with this concept, antioxidants that targeted mitochondrial oxidative stress have previously been documented to preserve the membrane potential of isolated mitochondria from lung tissues of mice chronically exposed to ozone and to reduce inflammation in airway smooth muscle cells from smokers and patients with chronic obstructive pulmonary disease (COPD)[45].

4.3. Targeted antioxidant therapies in chronic lung diseases

Oxidative stress is a hallmark of chronic exposure to cigarette smoke [6] and a critical driving mechanism in the pathogenesis of COPD [46], which has therefore provided the impetus for testing antioxidant therapies in these populations. However, the effects of dietary or thiol-based antioxidant at improving clinical outcomes in patients with COPD have been rather inconclusive [47–49]. Interestingly, pre-clinical evidence have suggested that targeted therapies might be more effective and have greater therapeutic potential. For instance, SOD mimetics (AEOL 10150) has shown encouraging results, diminishing the inflammatory response of the lungs in tobacco smoked-exposed mice [50]. Ebselen, a glutathione peroxidase mimetic, has been documented to reduce airway inflammation and inflammatory cytokines in mice exposed to cigarette smoke [51,52]. Although not an antioxidant per se, apocynin, which inhibit NADPH oxidase, attenuated the pro-inflammatory lung response induced by cigarette smoke exposure [53,54] and abolished the loss of muscle mass and function [53]. In the context of smoke-related conditions, mitochondria-targeted antioxidant treatments have been limited to in vitro and in situ experiments [47–49]. In humans, MitoQ, a derivative of ubiquinone conjugated to triphenylphosphonium, has also been shown to improve arterial function in older adults and reduced oxidative stress markers in chronic hepatitis C patients (Rossman et al., 2018; Schulz et al., 2010). Overall, despite promising pre-clinical findings, there is a crucial lack of clinical trials specifically examining the therapeutic effectiveness of these targeted antioxidant strategies on physiological function and clinical outcomes in smoke-related conditions.

4.4. Experimental consideration

The current experimental model in situ offers the opportunity to investigate the acute effects of CSC exposure on mitochondrial function within a normal cellular architecture preserving mitochondrial morphology and interactions with other structures (cytoskeleton, nucleus, endoplasmic reticulum). The advantage of this model is to provides under rigorously controlled condition some mechanistic insights on the initial metabolic insults caused by CSC to identify sites within the oxidative phosphorylation that may be more sensitive to CSC toxicity, and therefore potential candidates for therapeutics with chronic exposure. There are also limitations to this model as only the lipid soluble components of CS can be investigated, and the concentrations of CSC used in the present study reflect the level of exposure experienced by heavy smokers [55]. Also, this model is restricted to short-term exposure [17] as mitochondrial functional integrity becomes compromised after ~30 h of cold storage [56,57].

This study did not comprehensively evaluate MitoTEMPO dose-response in CSC exposed muscle fibers. We acknowledge the importance of characterizing MitoTEMPO pharmacokinetics in situ and in vivo for future clinical studies in people chronically exposed to secondhand cigarette smoke or patients with COPD. However, our primary objective in this study was to determine whether mitochondrial ROS production played a role in CSC induced metabolic dysregulation and mitochondrial metabolism, which is a necessary first step to identify potential cellular targets for mitigating the deleterious effects of smoking. Another limitation of the present study was the lack of assessment of enzymatic activities in tissue homogenates, which could have provided some additional information on the CSC-induced change in activity of some specific enzymes of the TCA cycle (e.g. pyruvate dehydrogenase, aconitase).

While our study focuses on the impact of cigarette smoke condensate and MitoTEMPO treatment on mitochondrial respiration, it should be noted that we did not directly measure free radical production due to the technical challenges of directly and accurately quantifying ROS generation in permeabilized tissues under different metabolic states. This limitation leaves some open questions about the specific sites of mitochondrial ROS production induced by CSC. Future research, perhaps, combining metabolically active permeabilized tissues to direct free radical measurement by electron paramagnetic resonance (EPR) spectroscopy, will be essential to better pinpoint the predominant sites involved in this vicious cycle that promotes oxidative stress and aggravates bioenergetic deficits in the mitochondria exposed to CSC.

We selected the fast-twitch gastrocnemius muscle based on the known effect of cigarette smoke exposure in shifting muscle fiber type toward fast twitch glycolytic fibers [58]. In addition, previous work from our group [16–19], suggested that, other than different susceptibilities to CSC, the fundamental mechanisms involved in CSC inhibition of mitochondrial respiration were similar between fast and slow-twitch muscle fibers. It is, however, noteworthy that slow-twitch oxidative fibers exhibit a greater antioxidant capacity. Therefore, future research is warranted to include evaluations of the effects of MitoTEMPO on mitochondrial respiration in various skeletal muscles, such as the diaphragm and intercostal muscles, which play important roles in respiratory function and exhibit a slow-twitch muscle phenotype. Additionally, given the importance of bronchial smooth muscle cells in the pathophysiology of COPD, specific studies on these cells are required. Bronchial smooth muscle cells are directly exposed to inhaled cigarette smoke and are crucial in airway remodeling and hyperresponsiveness observed in COPD. Investigating the effects of Mito-TEMPO on mitochondrial function and oxidative stress in bronchial smooth muscle cells will further our understanding of its potential therapeutic role in COPD.

It is also important to note that CSC and MT were administered concurrently in the present study. Therefore, this treatment may be less effective in conditions where oxidative damage is advanced or irreversible such as severe to very severe COPD patients, and may be better suited for middle-aged individuals exposed to ciagrette smoke or COPD patients at an early stage of the disease. This highlights the need for further studies to investigate the isolated and combined effects of MT in the absence of preventative interventions to better understand its standalone efficacy in treating smoke-related conditions.

4.5. Clinical implications

Patients with COPD, a disease primarily caused by cigarette smoking, frequently experience exaggerated lactate accumulation and muscle acidosis during exercise [59–61]. Interestingly, previous studies also documented greater pyruvate levels coupled to unaltered pyruvate dehydrogenase activity in the vastus lateralis muscle of patients with COPD compared with age-matched healthy controls [62,63]. Conceptually, the accumulation of pyruvate in the cytosol may be due to pyruvate carrier inhibition and/or slower reaction rates through the TCA cycle, ultimately leading to the conversion of pyruvate into lactate. Our findings lend support to this hypothesis and suggest that lactate accumulation during exercise is likely a compensatory mechanism to maintain ATP supply caused by impaired oxidative phosphorylation capacity in the skeletal muscle of chronic smokers and patients with COPD. Restoring mitochondrial function through physical rehabilitation [64–66] and/or mitochondria-targeted antioxidant therapies may thus have some implications both in term of metabolic homeostasis and redox signaling for patients with COPD. The beneficial effects of MitoTEMPO on maximal ADP-stimulated respiration in the gastrocnemius muscle exposed to CSC acutely indicates that mitochondrial redox balance may be a potential therapeutic target to improve exercise tolerance and quality of life in patients with COPD.

Inhaled triple therapy, comprising inhaled corticosteroids (ICS), long-acting beta-agonists (LABA), and long-acting muscarinic antagonists (LAMAs), is the current gold standard for COPD treatment [67]. Adding antioxidant agents, such as MitoTEMPO, to this regimen could provide additional therapeutic benefits by targeting mitochondrial oxidative stress, which is a key contributor to COPD pathogenesis. However, integrating antioxidants into existing treatment protocols poses several challenges, including determining optimal dosages, ensuring targeted delivery to affected tissues, and assessing long-term safety and efficacy.

5. Conclusion

In conclusion, this study revealed that MitoTEMPO attenuated cigarette smoke condensate-induced inhibition of mitochondrial respiration in permeabilized muscle fibers of middle-aged mice. MitoTEMPO treatment improved, but did not fully restore, both the maximal muscle respiratory capacity and complexes I and II specific respiration. The sequential utilization of pyruvate- and glutamate-stimulated respiration revealed a rate-limiting step in the TCA cycle upstream of α-ketoglutarate, implicating the respective transporters and enzymes preceding α-ketoglutarate metabolism. These findings, in fast-twitch gastrocnemius muscle fibers from middle-aged mice, highlight the role of mitochondria as a target of cigarette smoke toxicity, but also a causal factor to tissue and organ dysfunction by accentuating oxidative stress and metabolic disturbance related to cigarette smoke exposure. Cumulatively, these findings shed light on the metabolic alterations associated with smoking and offer a potential target for future therapeutics in patients with COPD.

Data availability

Data are available upon reasonable request by contacting glayec@unomaha.edu.