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Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria Wieslawa Jarmuszkiewicz, Andrzej WoydaPloszczyca, Agnieszka Koziel, Joanna Majerczak, Jerzy A. Zoladz www.elsevier.com/locate/freeradbiomed
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S0891-5849(15)00075-1 http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.012 FRB12314
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Free Radical Biology and Medicine
Received date: 1 October 2014 Revised date: 5 February 2015 Accepted date: 8 February 2015 Cite this article as: Wieslawa Jarmuszkiewicz, Andrzej Woyda-Ploszczyca, Agnieszka Koziel, Joanna Majerczak, Jerzy A. Zoladz, Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria Wieslawa Jarmuszkiewicz1*, Andrzej Woyda-Ploszczyca1#, Agnieszka Koziel1#, Joanna Majerczak2 and Jerzy A. Zoladz2 1
From the Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland; 2Department of Muscle Physiology, Chair of Physiology and Biochemistry, Faculty of Rehabilitation, University School of Physical Education, Krakow, Poland Running title: Effect of temperature on muscle mitochondrial function *
To whom correspondence should be addressed: Wieslawa Jarmuszkiewicz, Department of Bioenergetics, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland, Tel.: (48-61) 8295881; Fax: (48-61) 8295636; Email: [email protected] #
These authors contributed equally to this work.
Keywords: skeletal muscle mitochondria; temperature; oxidative phosphorylation; uncoupling protein; mitochondrial function Abbreviations: BSA, bovine serum albumin; CS, citrate synthase; COX, cytochrome c oxidase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OXPHOS, oxidative phosphorylation; RCR, respiratory control ratio; ROS, reactive oxygen species; Q, quinone, coenzyme Q; UCP, uncoupling protein; ∆Ψm, mitochondrial transmembrane electrical potential Highlights •
Increase in temperature augments cytochrome oxidase and citrate synthase activities.
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Temperature enhancement reduces membrane potential and quinone reduction levels.
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Temperature increase decreases mitochondrial ROS production and OXPHOS efficiency.
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Temperature-induced proton leak influences ROS production and OXPHOS efficiency.
Abstract Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25ºC) and hyperthermia (42ºC) compared to the physiological temperature of resting muscle (35ºC). For non-phosphorylating mitochondria, increasing the temperature from 25ºC to 42ºC led to a decrease in membrane potential, hydrogen peroxide production and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were
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observed. However, the efficiency of oxidative phosphorylation decreased, while the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased with increasing assay temperature. An increase in proton leak, including uncoupling proteinmediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production.
Introduction Mitochondrial oxidative phosphorylation (OXPHOS) plays a key role in skeletal muscle energy homeostasis under various physiological conditions (see e.g. Hochachka 1994; Blomstrand et al. 1997; Korzeniewski & Zoladz 2003, Zoladz et al. 2006; Jones et al. 2011). However, both the rate of this metabolic process and its efficiency in skeletal muscle can be significantly affected by changes in muscle temperature (Brooks et al. 1971; Willis & Jackman 1994), which at rest amounts to 35-36°C in rats (Brooks et al. 1971) and humans (Saltin et al. 1968; Beelen & Sargeant 1991). Various strains of rats can survive a decrease in their core temperature to approximately 15oC and its increase to approximately 43oC (Musacchia & Jacobs 1973; Furuyame 1982). Interestingly, similar values of the highest and lowest survivable core temperatures have been reported for humans (Bynum et al. 1978; Tsuei & Kearney 2004). In rat skeletal muscle mitochondria, with increasing assay temperature (from 37oC to 43-45°C), the respiratory acceleration and a pronounced decrease in ADP/O ratio have been observed (Brooks et al. 1971; Willis & Jackman 1994). With decreasing assay temperature (from 37°C to 25°C), a significant decrease in respiratory rates has been accompanied by no change in ADP/O ratio (Brooks et al. 1971). Surprisingly, the effect of the extremely high and very low temperatures on the function of skeletal muscle mitochondria, especially their phosphorylation activities, uncoupling and reactive oxygen species (ROS) production is still poorly understood. It is well documented that mitochondria produce ROS, which are generated as a byproduct of oxygen metabolism (for review see Packer et al. 2008; Murphy et al. 2009; Kowaltowski et al. 2009; Figueira et al. 2013; Quinlan et al. 2013). The enhanced ROS production seems to be involved in cell damage. On the other hand, there is a growing body of evidence that the enhanced ROS production stimulates vital processes for maintaining muscle homeostasis and muscle adaptation to exercise including mitochondria biogenesis (St-Pierre et al. 2006; Jackson 2008; Powers et al. 2011; Austin et al. 2012; Halliwell 2012). Interestingly, in rat brain mitochondria, a decrease in temperature from 37°C to 32°C results in an increase in ROS production (Ali et al. 2010). Surprisingly, to the best of our knowledge, no effect of 2
hyperthermia or hypothermia on skeletal muscle mitochondrial ROS production has been studied so far. Similarly, the literature poorly documents on the role of mitochondrial membranes (via the proton conductance) in the control of OXPHOS by varying the temperature. Mitochondrial energy metabolism, especially the OXPHOS step, is strictly controlled by a proton electrochemical gradient resulting from oxidation of reducing fuels and from a proton leakage through the inner mitochondrial membrane. Similarly, mitochondrial ROS production is described to be a direct function of the proton electrochemical gradient and controlled by mild uncoupling via proton leakage (Kowaltowski et al. 2009). These processes should be extremely important in skeletal muscle mitochondria that produce a significant amount of ATP in the mammalian body and that undergo physiological changes in surrounding tissue temperature. Therefore, in the present study, our aim was to establish the effect of experimentally induced hyperthermia (42°C) and hypothermia (25°C) on the function of isolated rat skeletal muscle mitochondria. Particularly, respiratory rate activities under phosphorylating and non-phosphorylating conditions, the yield of ATP synthesis, and for the first time, mitochondrial membrane potential (∆Ψm), mitochondrial uncoupling (proton leakage) and ROS production were studied in temperatures of 25°C, 35°C and 42°C. Moreover, the oxidative capacity of rat skeletal muscle mitochondria was studied for the first time under the tested temperatures by measuring maximal activities of cytochrome c oxidase (COX) and citrate synthase (CS). The novelty of the present study lies in demonstrating that increase in proton leak, resulting from increasing assay temperature could account for the reduced OXPHOS efficiency and ROS production.
Material and methods Chemicals All chemicals were the highest available grades and were purchased from Sigma-Aldrich unless otherwise mentioned.
Animals The experiments were carried out on adult 4-6-month-old male Wistar rats weighing 475530 g. The animals were bred in the animal house at the Poznan University of Medical Sciences, Poznan, Poland. They were given free access to water and pellet food and were housed under standard humidity and temperature conditions on a 12 h light/dark cycle. Experimental protocols involving animals, their surgery and care were approved by the Local 3
Ethics Committee on Animal Experimentation in Poznan, Poland and were in compliance with the guidelines of the European Community Council Directive on the protection of animals used for scientific purposes. Animals were sacrificed, and all efforts were made to minimize suffering.
Tissue preparation and mitochondria isolation The gastrocnemius, soleus, and quadriceps muscles were quickly excised and placed into an ice-cold isolation medium that contained 100 mM sucrose, 100 mM KCl, 50 mM TrisHCl, 1 mM KH2PO4, 0.5 mM EDTA, 0.1 mM EGTA and 0.2% bovine serum albumin (BSA), pH 7.2. All subsequent steps were performed at 4°C. The excised muscles were trimmed to remove adipose and connective tissue, finely minced and homogenized. First, muscle portions were homogenized using a Polytron homogenizer (T18 basic, IKA) (three times for 2 s, at 80% power) and then by four passes with a glass-Teflon homogenizer. The homogenate was filtered through a double layer of cheesecloth and centrifuged at 700 g for 10 min. The resultant supernatant was filtered again and then centrifuged at 9,000 g for 10 min to pellet mitochondria. The mitochondrial pellet was then resuspended in the isolation medium without BSA and centrifuged at 700 g for 10 min. The supernatant was filtered and centrifuged at 9,000 g for 10 min. The final mitochondrial pellet was then suspended in a small volume (11.5 ml) of a suspension buffer containing 225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA and 10 mM Tris-HCl, pH 7.2. Mitochondrial protein concentration was determined by the by the Bradford method with BSA as a standard (Bradford 1976).
Maximal citrate synthase (CS) and cytochrome c oxidase (COX) activities The maximal CS and COX activities were assayed at various temperatures (25°C, 35°C and 42°C) as described previously (Zoladz et al. 2014). CS activity was measured with a UV1650 Shimadzu spectrophotometer at 412 nm with 100 mM 5,5’-di-thiobis-(2-nitrobenzoic acid) (TNB) and 40-60 µg of muscle mitochondrial protein. CS activity was expressed as nmol TNB x min-1 x mg protein-1. COX activity was measured polarographically using a Clark-type oxygen electrode (Hansatech) in 1 ml of a respiration medium containing 225 mM mannitol, 75 mM sucrose, 10 mM KCl, 5 mM KH2PO4, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% BSA, 10 mM Tris-HCl, pH 7.2, with 0.1-0.12 mg of muscle mitochondrial protein. COX activity was assessed in the presence of sequentially added antimycin A (10 ȝM), 8 mM ascorbate, 0.06% cytochrome c, and up to 1 mM (N,N,N’N’-tetramethyl-p-phenylenediamine (TMPD). COX activity was expressed in nmol O x min-1 x mg protein-1. 4
Measurements of mitochondrial respiration and membrane potential Measurements were performed at various temperatures (25°C, 35°C and 42°C). Oxygen uptake was determined polarographically using a Rank Bros. oxygen electrode or a Hansatech oxygen electrode in either 2.8 ml or 0.7 ml of the respiration medium (see above), with either 1 or 0.25 mg of mitochondrial protein (0.36 mg/ml). The temperature of the electrode chambers was tightly controlled with Lauda Ecoline 003 thermostats. Oxygen electrodes were calibrated after each change in assay temperature. ǻȌm was measured simultaneously with oxygen uptake using a tetraphenyl-phosphonium (TPP+)-specific electrode. The TPP+electrode was calibrated after each change in assay temperature by four sequential additions (0.4, 0.4, 0.8, and 1.6 µM) of TPP+. After each run, 0.5 µM carbonyl cyanide ptrifluoromethoxyphenyl-hydrazone (FCCP) was added to release TPP+ for base-line correction. For the calculation of the ǻȌ value, the matrix volume of skeletal muscle mitochondria was assumed to be 2.0 ȝl × mg−1 protein. The values of ∆Ψm were corrected for TPP+ binding using the apparent external and internal partition coefficients of TPP+ (WoydaPloszczyca & Jarmuszkiewicz 2011). The correction shifted the calculated ∆Ψm values to lower values (approx. 30 mV-shift), but did not influence the changes in the resulting membrane potential (relative changes). The values of ǻȌm were given in mV. Succinate (5 mM) with rotenone (2 µM) or malate (5 mM) plus pyruvate (5 mM) were used as respiratory substrates. OXPHOS studies were performed in the absence of Mg2+ to avoid the adenine nucleotide interconversion catalyzed by mitochondrial adenylate kinase. Phosphorylating respiration (state 3) was measured after an ADP pre-pulse (50 µM) using 150 µM ADP as a main pulse. The total amount of oxygen consumed during state 3 respiration was used for calculation of the ADP/O ratio. Measurements of ∆Ψm allowed for fine control of the duration of state 3 respiration. The proton-conductance response to a driving force can be expressed as the relationship between the oxygen consumption rate and the ∆Ψm (flux/force relationship) when varying the potential by titrating with respiratory-chain inhibitors. Proton leak assessments during nonphosphorylating (resting, state 4) respiration were performed as previously described (SwidaBarteczka et al. 2009) with 5 mM succinate (plus 2 µM rotenone) as an oxidizable substrate in the absence of exogenous ADP and the presence of 1.8 ȝM carboxyatractyloside and 0.7 µg/ml (2 µg per mg of protein) oligomycin, which inhibited the activities of an ATP/ADP antiporter and ATP synthase, respectively. MgCl2 (0.5 mM) was added to the respiration
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medium (see above). To induce uncoupling protein (UCP2/3) activity, linoleic acid (up to 16 µM) was used. To inhibit UCP2/3 activity, 2 mM GTP was applied. To decrease the rate of the Q-reducing pathway, succinate dehydrogenase was titrated with malonate (up to 1.7 mM).
Assay of H2O2 production by isolated mitochondria Mitochondrial H2O2 production was measured by the Amplex Red-horseradish peroxidase method (Invitrogen) (Zhou et al. 1997). Horseradish peroxidase (0.1 units/ml) catalyzes the H2O2-dependent oxidation of nonfluorescent Amplex Red (5 µM) to fluorescent resorufin red. Fluorescence was kinetically followed for 15 min at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a Infinite M200 PRO Tecan multimode reader that was adjusted internally to different temperatures. Mitochondria (0.08-0.1 mg of mitochondrial protein) were incubated in 0.5 ml of the respiration medium (see above) with 5 mM succinate as an oxidizable substrate in the absence or presence of rotenone (2 µM), oligomycin (0.7 µg/ml), carboxyatractyloside (1.8 µM) or 0.5 mM MgCl2. Reactions were monitored with constant stirring and calibrated with known amounts of H2O2. H2O2 production rates were determined from slopes calculated from readings obtained along several 15-min repeated measurements.
The determination of quinone (Q) reduction level The reduced Q versus the total endogenous pool of Q in the inner mitochondrial membrane was determined by an extraction technique followed by HPLC measurements (Jarmuszkiewicz et al. 2004; Swida et al. 2008). For calibration of the HPLC peaks, commercial Q9 was used (Sigma-Aldrich cat. no. 27597).
Statistical analysis The results are presented as the means ± S.D. obtained from 6-13 independent mitochondrial isolations, in which each determination was performed at least in duplicate. An unpaired two-tailed Student’s t-test was used to identify significant differences; in particular, differences were considered to be statistically significant if p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).
Results
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Mitochondrial oxidative capacities increased under hyperthermia and decreased under hypothermia The oxidative capacities were elucidated by measuring the maximal COX and CS activities at various temperatures (Fig. 1). When the temperature of the incubation medium was increased from 35ºC to 42ºC, a value to which mammalian muscle temperature can increase during exercise (Saltin et al. 1968), activities of both enzymes significantly enlarged by approximately 56% and 26% for COX and CS, respectively (Fig. 1). Conversely, under hypothermia conditions, when the assay temperature was decreased from 35ºC to 25ºC, a significant reduction by approximately 32% and 21% of COX and CS activities, respectively, was observed. Thus, in rat muscle mitochondria, the oxidative capacities of the respiratory chain and TCA cycle in parallel react to changes in external temperature with both being increased or decreased under hyperthermia or hypothermia conditions, respectively.
The increase in assay temperature augmented the oxidation and phosphorylation rates but decreased the efficiency of oxidative phosphorylation The different parameters of OXPHOS were studied as a function of temperature (Fig. 2, Table 1). For both applied respiratory substrates (succinate+rotenone and malate+pyruvate), the considerable increase in the oxidation rate with the increasing temperature was observed both in state 3 and in state 4 (Fig. 2A, Table 1). Namely, the increase in measurement temperature from 25°C to 42°C resulted in an approximately five-fold increase in the rate of non-phosphorylating respiration and an approximately three-fold increase in phosphorylating respiration. Figure 2B shows that, following oxidation rate, ATP synthesis is also elevated significantly (approximately two-fold) for both applied substrates. In contrast to the oxidation and phosphorylation rates, the OXPHOS efficiency (ADP/O ratio) and the respiratory control ratio (RCR) decreased significantly with the increasing temperature (Fig. 2C, D, Table 1), indicating a possible uncoupling. For succinate+rotenone, between 25°C and 42°C, the ADP/O ratio reduced from approximately 1.4 to 1.05 and the RCR reduced from approximately 5 to 3. For malate+pyruvate, the ADP/O ratio reduced from approximately 2.4 to 1.8, and the RCR reduced from approximately 6 to 3.7 with a temperature increase from 25°C to 42°C. Thus, although the oxidation and phosphorylation activities increased with temperature, the highest apparent ADP/O ratio and RCR were observed at 25°C, far below the physiological value.
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The ∆Ψm value, which is maximal under non-phosphorylating conditions and decreases under ADP phosphorylation, reflects the substrate oxidation (proton electrochemical gradient producing reactions), and ATP turnover and proton leak (proton electrochemical gradient consuming reactions). With an increasing temperature, no temperature-dependent statistically significant changes in ∆Ψm values were observed for phosphorylating mitochondria for both studied respiratory substrates (Fig. 2E, Table 1). However, the increase in assay temperature from 25°C to 42°C resulted in a significant decrease in ∆Ψm in state 4; for succinate+rotenone, the decrease was from approximately 174 mV to 167 mV and for malate+pyruvate, the decrease was from approximately 162 mV to 156 mV. These changes were accompanied by a significant decrease in depolarization (change in ∆Ψm in response to ADP phosphorylation) from approximately 33 mV to 23 mV and from approximately 22 mV to 15 mV, for succinate+rotenone and malate+pyruvate, respectively. Thus, in rat skeletal muscle mitochondria, the lower ∆Ψm values of non-phosphorylating respiration and the decreased depolarization during the state 4 to state 3 transition, strongly suggest that the lower ADP/O and RCR ratios are the consequence of mitochondrial uncoupling increasing together with surrounding temperature.
The ATP turnover-independent proton leakage increased with the increasing assay temperature A possible modification of proton leakage was suggested by the OXPHOS studies discussed above. The proton electrochemical gradient (with ∆Ψm as the main component) is produced by reducing substrate oxidation and consumed by ATP synthesis (ADP phosphorylation) and ATP turnover-independent proton leakage. Proton leakage decreases the OXPHOS efficiency. Figure 3 shows proton conductance kinetics (the relationships between oxygen uptake and ∆Ψm obtained during progressive inhibition of the respiratory chain) in the absence or presence of OXPHOS inhibitors (oligomycin and carboxyatractyloside) for the three tested temperatures. In the absence of added ADP and OXPHOS inhibitors, respiration involves proton leak-driven respiration and endogenous ATP turnover-driven respiration. In the presence of OXPHOS inhibitors, proton leak kinetics can be analyzed. The non-ohmic curves for proton conductance were obtained for temperatures of 25°C, 35°C and 42°C. For all temperatures, addition of OXPHOS inhibitors shifted the proton conductance kinetic curves to the higher ∆Ψm and lower respiratory rate values, indicating the exclusion of ATP turnover-linked conductance. Namely, approximately 6 mV, 7 mV and 9 mV of OXPHOS
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inhibitor-induced increases in ∆Ψm values were observed for initial, uninhibited respiratory rates for 25°C, 35°C and 45°C, respectively (Fig. 3A, B). With increasing assay temperature, the relationships between oxidation rate and ∆Ψm were shifted to the lower ∆Ψm and higher respiratory rate values, both in the absence or presence of OXPHOS inhibitors (Fig. 3A, B). Moreover, at a higher measurement temperature, a steeper relationship between oxidation rate and ∆Ψm was observed. Figure 3C presents respiratory rates measured at common ∆Ψm (157 mV) for all tested temperatures in the absence (proton leak-driven respiration and ATP turnover-driven respiration) or presence (proton leak-driven respiration) of OXPHOS inhibitors. The results indicate that the ATP turnover-independent proton leakage, together with the ATP turnover-linked proton conductance, increased with increasing assay temperature from 25°C to 42°C in rat skeletal muscle mitochondria.
The increase in assay temperature increased UCP-mediated proton leakage To exclude inducible fatty acid-mediated leak through ATP/ADP antiporter, all measurements of UCP-mediated uncoupling were performed in the presence of carboxyatractiloside. Unlike the ATP/ADP antiporter-mediated proton conductance, which can be inhibited by GDP, UCPs are strongly inhibited by GTP, in addition to GDP (WoydaPloszczyca & Jarmuszkiewicz 2014). Because, the inhibition of mitochondrial proton conductance by GTP but not GDP should be considered diagnostic of UCP function, we used GTP to determine UCP activity in isolated rat skeletal muscle mitochondria (Fig. 4 , 6). To investigate UCP-mediated proton conductance, the flux-force relationships were obtained in the presence of a low concentration (16 µM) of linoleic acid and 2 mM GTP in non-phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C (Fig. 4). In general, kinetic curves of linoleic acid-induced GTP-inhibited proton leak (UCP activity) were shifted to the lower ∆Ψm and higher respiratory rate values with increasing measurement temperature (Fig. 4A). At a higher temperature, linoleic acid induced a more pronounced mitochondrial uncoupling (a stronger increase in oxygen consumption and a stronger decrease in ∆Ψm), while GTP caused a greater inhibition of this uncoupling (a stronger decrease in the oxidation rate and a stronger increase in ∆Ψm). At the highest common ∆Ψm value (162 mV), between 25°C and 45°C, the linoleic acid-induced proton leak and the linoleic acid-induced GTP-inhibited proton leak increased approximately four
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times (Fig. 4B). Thus, we can conclude that in rat skeletal muscle mitochondria, UCPmediated uncoupling elevated with increasing assay temperature.
At a given temperature, mitochondrial ROS production lowered under the phosphorylating and uncoupling conditions Figure 5A shows a comparison of H2O2 production of isolated rat skeletal muscle mitochondria under non-phosphorylating and phosphorylating conditions. Because with malate-pyruvate (complex I substrates), an approximately 2.4 times lower H2O2 production rate was found (at a given temperature) compared to succinate (complex II substrate), results for phosphorylating mitochondria are presented only with succinate. At all tested assay temperatures (25°C, 35°C and 42°C), rates of H2O2 production during succinate oxidation were elevated approximately 2.5-4.5 times in resting state 4 compared to phosphorylating state 3. Mitochondrial ROS production was higher under state 4 conditions, because ∆Ψm across the inner mitochondrial membrane (Fig. 2E) and the Q reduction level (Fig. 5B) were high. At all tested assay temperatures, H2O2 production by succinate-oxidizing nonphosphorylating mitochondria was approximately 32-37% lower in the presence of rotenone compared to conditions without the inhibitor of complex I (Fig. 5C). Therefore, measurements of H2O2 production in phosphorylating mitochondria were performed in the absence of rotenone (Fig. 5A). Mitochondrial ROS production is thought to be a direct function of the proton electrochemical potential and to be controlled by mild uncoupling via proton leakage through the inner mitochondrial membrane (Kowaltowski et al. 2009). Therefore, at all tested assay temperatures, in the presence of MgCl2 (which inhibits proton conductance in rat skeletal muscle mitochondria) (Cadenas & Brand 2000), oligomycin and carboxyatractyloside (which inhibit the ATP turnover-linked H+ leak), a 6-8 times higher H2O2 production was observed under non-phosphorylating conditions compared to state 4 without these additions (Fig. 5C). Higher H2O2 production in the presence of MgCl2, oligomycin and carboxyatractyloside indicates the exclusion of ATP turnover-linked uncoupling (H+ leak). Moreover, at all tested assay temperatures, a low concentration of linoleic acid (10 µM) decreased H2O2 production, while the subsequent addition of GTP (2 mM) partially restored H2O2 production (Fig. 6A). The free fatty acid-induced purine nucleotide-inhibited change in H2O2 production can be attributed to the UCP-mediated uncoupling (H+ leak) that leads to a stimulation of respiration,
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partial ∆Ψm depolarization and decrease in Q reduction level (Fig. 4) (Jarmuszkiewicz et al. 2004 for Q reduction level). Thus, we can conclude that in rat skeletal muscle mitochondria, at a given assay temperature, ROS production decreased both under phosphorylating conditions and under uncoupling conditions (with the ATP turnover-linked and UCP-mediated leakages), and thus, at lowered ∆Ψm and Q reduction level.
Under non-phosphorylating conditions, mitochondrial ROS production and Q reduction level reduced with the increasing assay temperature When analyzing the effect of temperature on ROS production in non-phosphorylating mitochondria, a significant increase in H2O2 production (and Q reduction level) at a lower temperature (25ºC) and a significant decrease in H2O2 production (and Q reduction level) at a higher temperature (42ºC) compared to control conditions (35ºC) were observed with succinate and malate+pyruvate as respiratory substrates (Fig. 5A, B). This temperature dependence was observed in non-phosphorylating (state 4) succinate-oxidizing mitochondria both in the absence and presence of rotenone, MgCl2, oligomycin, carboxyatractyloside (Fig. 5) or linoleic acid (Fig. 6) thus independent of the ATP turnover-linked and UCP-mediated leakages. However, no temperature-dependent statistically significant changes in H2O2 production and Q reduction level were observed for phosphorylating mitochondria (state 3) (Fig. 5A, B). Thus, the increase in temperature from 25ºC to 42ºC led to the reduction in ROS production only under non-phosphorylating conditions that was accompanied by a significant decrease in Q reduction level (Fig. 5B) and state 4/ state 3 H2O2 production ratio (from approximately 4.5 to 2.5) (Fig. 5A). Moreover, the linoleic acid-induced GTP-inhibited changes in H2O2 production, which can be attributed to the UCP activity, elevated with the increase in the temperature from 25ºC to 42ºC (Fig. 6B). Thus, we can conclude that under non-phosphorylating conditions independent of the ATP turnover-linked and UCP-mediated leakages, mitochondrial ROS production and Q reduction level diminished with the increasing assay temperature.
Discussion For the first time, the role of the inner membrane conductance to protons in the mitochondrial OXPHOS process and mitochondrial ROS production was studied in skeletal
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muscle mitochondria during experimentally induced hypothermia (25ºC) and hyperthermia (42ºC) compared to the physiological temperature of resting muscle (35ºC). For nonphosphorylating skeletal muscle mitochondria, increasing the temperature from 25ºC to 42ºC led to a decrease in ∆Ψm, H2O2 production and Q reduction level (Figs. 2 and 5, Table 1). For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. Moreover, the efficiency of OXPHOS decreased, while the oxidation and phosphorylation rates and oxidative capacities of rat skeletal muscle mitochondria increased with increasing assay temperature (Figs. 1 and 2, Table 1). We found a significant decrease (by ~20%) in the ADP/O ratio and an acceleration of respiratory rates (state 3 by ~50% and state 4 by ~100%) and phosphorylation rate (by ~20-35%) in response to an increase in temperature of mitochondria from 35°C to 42°C (Table 1, Fig. 2). With increasing assay temperature, accelerated oxidation rate, resulting from increased proton leak, leads to accelerated ADP phosphorylation rate (respiration x ADP/O ratio) despite a decrease in the efficiency of OXPHOS (ADP/O ratio). The respiratory acceleration and a pronounced decrease in ADP/O ratio with increasing assay temperature have been previously observed with rat skeletal muscle mitochondria and rat liver mitochondria exposed to various temperatures (Brooks et al. 1971; Willis & Jackman 1994; Dufour et al. 1996). Namely, in rat skeletal muscle mitochondria, an increase in measurement temperature from 37°C to 43-45°C result in a significant increase in the phosphorylating (state 3) and non-phosphorylating (state 4) respiratory rates and in a decrease in ADP/O ratio (Brooks et al. 1971, Willis and Jackman 1994). An opposite effect, i.e., a pronounced decrease in respiratory rates has been observed during decreasing temperature from 37°C to 25°C, while ADP/O ratio remains unchanged (Brooks et al. 1971). In rat liver mitochondria, although oxidation and phosphorylation rates decrease with temperature (from 37°C to 4°C), the ADP/O ratio exhibits a maximum at 25°C (Dufour et al. 1996). Similarly to our results, in mice brain mitochondria, an increase in temperature from 32°C to 37°C resulted in respiratory acceleration, which was associated with a decrease in ROS production and attenuation of mitochondrial ∆Ψm (Ali et al. 2010). In rat skeletal muscle mitochondria, we demonstrated for the first time an increase in proton leak, including UCP-mediated proton leak (Fig. 4), with increasing assay temperature that could explain the lowering of OXPHOS efficiency and ROS production. However, one must remember that the increased temperature could also induce the leakiness of the inner mitochondrial membrane through membrane phospholipids or lipid-protein interactions (a basal proton conductance). The temperature increases could impact the integrity of the membrane, the embedded membrane proteins, the diffusion of molecules across the membrane and the activities
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of enzymes. In contrast to skeletal muscle mitochondria, in rat liver mitochondria, lack of
change in proton leakage (no increased uncoupling) between 25°C and 35°C does not account for the decrease in ADP/O ratio and RCR at the higher temperature (Dufour et al. 1996). Therefore, it seems that control of the mitochondrial proton leakage by temperature, which can influence the OXPHOS efficiency and ROS production level, depends on tissue. It could be at least partly related to the differences in the basal metabolic rates observed in various organs and tissues. It has been reported that the basal metabolic rate expressed per unit of mass in the liver (similar to its level in the brain) is approximately 20 times higher than in the skeletal muscle (Elia et al. 1992; Wang et al. 2012; Kummitha et al. 2014). Moreover, the results concerning the effect of hypothermia and hyperthermia on ADP/O ratio depend on the studied temperature range. In rat liver mitochondria exposed to varied temperatures in the range of 37°C to 4°C, a significant temperature dependence of ADP/O ratio has been observed with a maximum at 25°C (Dufour et al. 1996). In king penguin chick pectoralis muscle mitochondria, the ADP/O ratio was higher at 30°C than at 38°C (Monternier et al. 2014). The applied experimental procedure, i.e., the decreasing and increasing temperature of isolated mitochondria in relation to 35°C (a basal physiological temperature) is similar to cooling-down and warming-up of muscles in vivo by their exposure to the low or high temperature, respectively. In the present study, we demonstrated that an increase in temperature from 25°C to 42°C had a strong impact on maximal muscle COX and CS activities (Fig. 1A and B). It is worth noting that an elevation of temperature from 35°C to 42°C resulted in an 8% increase in COX activity and a 3.7% increase in CS activity per 1°C. An opposite effect, i.e., a decrease in the COX and CS activities (by 6.8% and by 3.2% per 1°C, respectively) was found when decreasing the temperature from 35°C to 25°C (Fig. 1A and B). This effect clearly shows that muscle mitochondrial OXPHOS capacity, as judged by the magnitude of maximal COX activity (Larsen et al. 2012; Zoladz et al. 2014), can be acutely modulated by changes in the muscle temperature. In the present study, we also determined the effect of varied temperatures on mitochondrial ROS production. We found 3-5-fold lower H2O2 production in state 3 compared to state 4 (Fig. 5A), indicating that H2O2 production in phosphorylating mitochondria is several times lower than in non-phosphorylating mitochondria. This is in agreement with the earlier studies showing that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation (Korshunov et al. 1997). Our results suggest that mitochondrial ROS production in working 13
muscles (likely with predominant phosphorylating mitochondria) is much lower than in muscles at rest (likely with predominant non-phosphorylating mitochondria). Moreover, in the present study we found that an increase in temperature from 25°C to 42°C had no effect on mitochondrial H2O2 production in state 3 and even decreased H2O2 production in state 4 (Fig. 5A). These results suggest that exposure of working muscles to high temperatures would not enhance mitochondrial ROS production, while exposure of non-working muscles to low temperatures would increase mitochondrial ROS production. Thus, our results suggest that the changes of muscle temperature might have different effects on mitochondrial ROS production in skeletal muscle at rest when compared to working muscle conditions.
Conclusions We found that temperature had a strong impact on the functional properties of skeletal muscle mitochondria. Increasing the assay temperature within the range of 25-42°C strongly increased the mitochondrial respiratory chain (including COX) and CS activities, resulting in an elevated phosphorylation rate. Moreover, temperature-induced decreases in OXPHOS efficiency and mitochondrial ROS production were observed, which may result from the temperature-induced increase in proton leak, including UCP-mediated proton leak. In skeletal muscle mitochondria, an increase in temperature from 25°C to 42°C decreased mitochondrial ROS production under non-phosphorylating conditions (likely predominant in resting muscle), while it had no effect on mitochondrial ROS production in phosphorylating mitochondria (likely predominant in working muscle).
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Musacchia XJ & Jacobs M (1973). Helium-cold induced hypothermia in the white rat. Proc. Soc. Exp. Biol. Med. 142, 734-739. Packer L, Cadenas E, Davies KJ (2008). Free radicals and exercise: an introduction. Free Radic. Biol. Med. 44, 123-125. Powers SK, Talbert EE & Adhihetty PJ (2011). Reactive oxygen and nitrogen species as intracellular signals in skeletal muscle. J. Physiol. 589, 2129-2138. Quinlan CL, Perevoshchikova IV, Hey-Mogensen M, Orr AL & Brand M.D. (2013). Sites of reactive oxygen species generation by mitochondria oxidizing different substrates. Redox Biol. 23, 304-312. Saltin B, Gagge AP & Stolwijk JA (1968). Muscle temperature during submaximal exercise in man. J. Appl. Physiol. 25, 679-688. St-Pierre J, Drori S, Uldry M, Silvaggi JM, Rhee J, Jager S, Handschin C, Zheng K, Lin J, Yang W, Simon DK, Bachoo R & Spiegelman BM (2006). Suppression of reactive oxygen species and neurodegeneration by the PGC-1 transcriptional coactivators. Cell 127, 397– 408. Swida-Barteczka A, Woyda-Ploszczyca A, Sluse F & Jarmuszkiewicz W (2009). Uncoupling protein 1 inhibition by purine nucleotides is under control of the endogenous ubiquinone redox state. Biochem. J. 424, 297-306. Swida A, Woyda-Ploszczyca A & Jarmuszkiewicz W (2008). Redox state of quinone affects sensitivity of Acanthamoeba castellanii uncoupling protein to purine nucleotides. Biochem. J. 413, 359-367. Tsuei BJ & Kearney PA (2004). Hypothermia in the trauma patient. Injury. 35, 7-15. Wang Z, Ying Z, Bosy-Westphal A, Zhang J, Heller M, Later W, Heymsfield SB & Müller MJ (2012). Evaluation of specific metabolic rates of major organs and tissues: comparison between nonobese and obese women. Obesity (Silver Spring) 20, 95-100. Willis WT & Jackman MR (1994). Mitochondrial function during heavy exercise. Med. Sci. Sports. Exerc. 26, 1347-1353. Woyda-Ploszczyca A & Jarmuszkiewicz W (2011). Ubiquinol (QH(2)) functions as a negative regulator of purine nucleotide inhibition of Acanthamoeba castellanii mitochondrial uncoupling protein. Biochim. Biophys. Acta 1807, 42-52. Woyda-Ploszczyca A & Jarmuszkiewicz W (2014). Different effects of guanine nucleotides (GDP and GTP) on protein-mediated mitochondrial proton leak. PLoS One 9, e98969. Zhou M, Diwu Z, Panchuk-Voloshina N & Haugland RP (1997). A stable nonfluorescent derivative of resorufin for the fluorometric determination of trace hydrogen peroxide: applications in detecting the activity of phagocyte NADPH oxidase and other oxidases. Anal. Biochem. 253, 162-168. Zoladz JA, Korzeniewski B & Grassi B (2006). Training-induced acceleration of oxygen uptake kinetics in skeletal muscle: the underlying mechanisms. J. Physiol. Pharmacol. 57 Suppl 10, 67-84. Zoladz JA, Grassi B, Majerczak J, Szkutnik Z, Korostynski M, Grandys M, Jarmuszkiewicz W & Korzeniewski B (2014). Mechanisms responsible for the acceleration of pulmonary V’O2 on-kinetics in humans after prolonged endurance training. Am. J. Physiol. 307, R1101-1114.
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Acknowledgements This work was supported by the grant of the Polish Ministry of Science and Higher Education (Harmonia 2013/08/M/NZ7/00787). We thank Malgorzata Budzinska and Wioletta Nobik for technical assistance.
FIGURE LEGENDS
Fig. 1. Maximal citrate synthase (CS) and cytochrome c oxidase (COX) activities of rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. The data are presented as the means ± S.D. (n = 15) and are from 5 independent mitochondrial preparations (triplicate assays for each condition). ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar) . Fig. 2. Temperature dependence of various energetic parameters with succinate or malate + pyruvate as respiratory substrates. (A) Respiratory rates were measured in the absence (state 4, st. 4) or presence (state 3, st. 3) of 150 µM ADP with 5 mM succinate (plus 2 µM rotenone) or 5 mM malate plus 5 mM pyruvate. The corresponding phosphorylation rate (B) and the respiratory control (C) and ADP/O ratios (D) are presented. The ADP phosphorylation rate is equal to state 3 respiration × ADP/O. The respiratory control ratio is equal to the ratio of state 3 to state 4 respiration. Membrane potential values (E) were measured under state 4 (∆Ψm4) and state 3 (∆Ψm3) conditions. The resulting ∆Ψm depolarization (∆Ψm4 minus ∆Ψm3) is also presented (F). (A-F) Every data point represents the mean ± S.D. (n = 18) for at least nine independent mitochondrial preparations, in which every condition was performed in duplicate. ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar). Fig. 3. Mitochondrial membrane conductance to protons at various temperatures. Respiratory rates and ∆Ψm were measured simultaneously during progressive inhibition by malonate (up to 1.7 mM) of the respiratory chain oxidizing succinate (5 mM) plus rotenone (2 µM) in the absence (A) or presence (B) of 0.7 µg/ml oligomycin (olig) and 1.8 µM carboxyatractyloside (CATR). (C) Respiratory rates at 157 mV in the absence or presence of oligomycin and carboxyatractyloside. Experiments were performed on mitochondria incubated at 25°C, 35°C or 42°C. The data are presented as the means ± S.D. (n = 15) and are from 5 independent mitochondrial preparations (triplicate assays for each conditions). ***, p < 0.001 versus values obtained at 35°C. Fig. 4. Kinetics of linoleic acid-induced, GTP-inhibited proton leak (UCP activity) in nonphosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. (A) Succinate (plus rotenone) oxidation was gradually decreased by increasing the concentration of malonate (up to 1.4 mM) in the presence of 0.7 µg/ml oligomycin (olig) and 1.8 µM carboxyatractyloside (CATR), in the absence or presence of 16 µM linoleic acid (LA) and in the absence or presence of 2 mM GTP. Data from representative experiment (mitochondrial preparation) are presented as the means ± S.D. (n = 3) (triplicate assays for each condition). Vertical lines indicate the linoleic acid-induced GTP-inhibited proton leak size at the highest common ∆Ψm value (at 162 mV) for all tested temperature conditions. (B) Values of the linoleic acid-induced proton leak and the linoleic acid-induced GTP-inhibited proton leak 17
obtained at 162 mV are presented. ***, p < 0.001, **, p < 0.01, versus values obtained at 35°C.
Fig. 5. H2O2 production (A, C) and Q reduction level (B) in non-phosphorylating and phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. (A, B) Measurements were performed with 5 mM succinate (in the absence of rotenone) or 5 mM malate plus 5 mM pyruvate in the absence (state 4, st. 4) or presence (state 3, st. 3) of 1 mM ADP. (A) The state 4/state 3 H2O2 production ratio is shown. (C) Effect of rotenone (2 ȝM) and oligomycin (olig, 0.7 µg/ml), carboxyatractyloside (CATR, 1.8 µM) and MgCl2 (0.5 mM) on H2O2 production of non-phosphorylating succinate-oxidizing mitochondria. (A, C) The data presented as the means ± S.D. (n = 24) are from 8 independent mitochondrial preparations (triplicate assays for each experiment). (B) The data (± S.D., n = 9) are from 3 independent mitochondrial preparations (triplicate assays for each experiment). ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar). Fig. 6. H2O2 production under linoleic acid-induced, GTP-inhibited proton leak (UCP activity) in non-phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. H2O2 production was measured under non-phosphorylating conditions with 5 mM succinate in the presence of oligomycin (olig, 0.7 µg/ml), carboxyatractyloside (CATR, 1.8 µM) and MgCl2 (0.5 mM). Linoleic acid (LA) (10 µM) and GTP (2 mM) were used to activate or inhibit the UCP activity, respectively. (B) The linoleic acid-induced changes in H2O2 production (difference in H2O2 production measured the presence of linoleic acid and in the absence of fatty acid) and the linoleic acid-induced GTP-inhibited changes in H2O2 production (difference in H2O2 production measured the presence of linoleic acid and in the presence of linoleic acid and GTP). (A, B) The data presented as the means ± S.D. (n = 24) are from 8 independent mitochondrial preparations (triplicate assays for each experiment). **, p < 0.01, *, p < 0.05 versus values obtained at 35°C.
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Table 1. Respiratory rates, ∆Ψm values and coupling parameters in rat skeletal muscle mitochondria at 25°C, 35°C and 42°C Experimental conditions are similar to those in Fig. 2. The respiratory rates (in nmol O x min1 x mg-1 protein) and ∆Ψm values (in mV) of state 3 (phosphorylating respiration) and state 4 (non-phosphorylating respiration following state 3) as well as corresponding respiratory control ratios (RCR) and ADP/O ratios are presented. Mean values (± S.D.) for at least nine different mitochondria preparations (with duplicate measurements, n = 18) are shown. ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a grey-shadowed column). malate + pyruvate 25°C 35°C 42°C State 3 197 ± 22*** 421 ± 45 609 ± 105* 141.7 ± 0.7 141.4 ± 2.2 ǻΨm3 140.5 ± 1.2 State 4 32.9 ± 3.4*** 84.9 ± 5.9 164 ± 13** ǻΨm4 162.2 ± 1.5* 159.4 ± 1.3 156.1 ± 2.0* RCR 5.98 ± 0.42* 4.96 ± 0.44 3.70 ± 0.2** ADP/O 2.43 ± 0.12 2.25 ± 0.16 1.79 ± 0.24** succinate + rotenone 25°C 35°C 42°C State 3 335 ± 32*** 602 ± 88 951 ± 61*** 143.2 ± 1.2 144.1 ± 1.4 ǻΨm3 141.0 ± 0.9 State 4 65.6 ± 6.1*** 150 ± 16 302 ± 35** 174.0 ± 1.9* 171.2 ± 0.7 167.4 ± 1.3* ǻΨm4 RCR 5.11 ± 0.24** 4.02 ± 0.34 3.15 ± 0.26** ADP/O 1.39 ± 0.06* 1.23 ± 0.08 1.06 ± 0.09*
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Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria Wieslawa Jarmuszkiewicz, Andrzej WoydaPloszczyca, Agnieszka Koziel, Joanna Majerczak, Jerzy A. Zoladz www.elsevier.com/locate/freeradbiomed
PII: DOI: Reference:
S0891-5849(15)00075-1 http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.012 FRB12314
To appear in:
Free Radical Biology and Medicine
Received date: 1 October 2014 Revised date: 5 February 2015 Accepted date: 8 February 2015 Cite this article as: Wieslawa Jarmuszkiewicz, Andrzej Woyda-Ploszczyca, Agnieszka Koziel, Joanna Majerczak, Jerzy A. Zoladz, Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria, Free Radical Biology and Medicine, http://dx.doi.org/10.1016/j.freeradbiomed.2015.02.012 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Temperature controls the oxidative phosphorylation and reactive oxygen species production through uncoupling in rat skeletal muscle mitochondria Wieslawa Jarmuszkiewicz1*, Andrzej Woyda-Ploszczyca1#, Agnieszka Koziel1#, Joanna Majerczak2 and Jerzy A. Zoladz2 1
From the Department of Bioenergetics, Faculty of Biology, Adam Mickiewicz University, Poznan, Poland; 2Department of Muscle Physiology, Chair of Physiology and Biochemistry, Faculty of Rehabilitation, University School of Physical Education, Krakow, Poland Running title: Effect of temperature on muscle mitochondrial function *
To whom correspondence should be addressed: Wieslawa Jarmuszkiewicz, Department of Bioenergetics, Adam Mickiewicz University, Umultowska 89, 61-614 Poznan, Poland, Tel.: (48-61) 8295881; Fax: (48-61) 8295636; Email: [email protected] #
These authors contributed equally to this work.
Keywords: skeletal muscle mitochondria; temperature; oxidative phosphorylation; uncoupling protein; mitochondrial function Abbreviations: BSA, bovine serum albumin; CS, citrate synthase; COX, cytochrome c oxidase; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone; OXPHOS, oxidative phosphorylation; RCR, respiratory control ratio; ROS, reactive oxygen species; Q, quinone, coenzyme Q; UCP, uncoupling protein; ∆Ψm, mitochondrial transmembrane electrical potential Highlights •
Increase in temperature augments cytochrome oxidase and citrate synthase activities.
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Temperature enhancement reduces membrane potential and quinone reduction levels.
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Temperature increase decreases mitochondrial ROS production and OXPHOS efficiency.
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Temperature-induced proton leak influences ROS production and OXPHOS efficiency.
Abstract Mitochondrial respiratory and phosphorylation activities, mitochondrial uncoupling and hydrogen peroxide formation were studied in isolated rat skeletal muscle mitochondria during experimentally induced hypothermia (25ºC) and hyperthermia (42ºC) compared to the physiological temperature of resting muscle (35ºC). For non-phosphorylating mitochondria, increasing the temperature from 25ºC to 42ºC led to a decrease in membrane potential, hydrogen peroxide production and quinone reduction levels. For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were
1
observed. However, the efficiency of oxidative phosphorylation decreased, while the oxidation and phosphorylation rates and oxidative capacities of the mitochondria increased with increasing assay temperature. An increase in proton leak, including uncoupling proteinmediated proton leak, was observed with increasing assay temperature, which could explain the reduced oxidative phosphorylation efficiency and reactive oxygen species production.
Introduction Mitochondrial oxidative phosphorylation (OXPHOS) plays a key role in skeletal muscle energy homeostasis under various physiological conditions (see e.g. Hochachka 1994; Blomstrand et al. 1997; Korzeniewski & Zoladz 2003, Zoladz et al. 2006; Jones et al. 2011). However, both the rate of this metabolic process and its efficiency in skeletal muscle can be significantly affected by changes in muscle temperature (Brooks et al. 1971; Willis & Jackman 1994), which at rest amounts to 35-36°C in rats (Brooks et al. 1971) and humans (Saltin et al. 1968; Beelen & Sargeant 1991). Various strains of rats can survive a decrease in their core temperature to approximately 15oC and its increase to approximately 43oC (Musacchia & Jacobs 1973; Furuyame 1982). Interestingly, similar values of the highest and lowest survivable core temperatures have been reported for humans (Bynum et al. 1978; Tsuei & Kearney 2004). In rat skeletal muscle mitochondria, with increasing assay temperature (from 37oC to 43-45°C), the respiratory acceleration and a pronounced decrease in ADP/O ratio have been observed (Brooks et al. 1971; Willis & Jackman 1994). With decreasing assay temperature (from 37°C to 25°C), a significant decrease in respiratory rates has been accompanied by no change in ADP/O ratio (Brooks et al. 1971). Surprisingly, the effect of the extremely high and very low temperatures on the function of skeletal muscle mitochondria, especially their phosphorylation activities, uncoupling and reactive oxygen species (ROS) production is still poorly understood. It is well documented that mitochondria produce ROS, which are generated as a byproduct of oxygen metabolism (for review see Packer et al. 2008; Murphy et al. 2009; Kowaltowski et al. 2009; Figueira et al. 2013; Quinlan et al. 2013). The enhanced ROS production seems to be involved in cell damage. On the other hand, there is a growing body of evidence that the enhanced ROS production stimulates vital processes for maintaining muscle homeostasis and muscle adaptation to exercise including mitochondria biogenesis (St-Pierre et al. 2006; Jackson 2008; Powers et al. 2011; Austin et al. 2012; Halliwell 2012). Interestingly, in rat brain mitochondria, a decrease in temperature from 37°C to 32°C results in an increase in ROS production (Ali et al. 2010). Surprisingly, to the best of our knowledge, no effect of 2
hyperthermia or hypothermia on skeletal muscle mitochondrial ROS production has been studied so far. Similarly, the literature poorly documents on the role of mitochondrial membranes (via the proton conductance) in the control of OXPHOS by varying the temperature. Mitochondrial energy metabolism, especially the OXPHOS step, is strictly controlled by a proton electrochemical gradient resulting from oxidation of reducing fuels and from a proton leakage through the inner mitochondrial membrane. Similarly, mitochondrial ROS production is described to be a direct function of the proton electrochemical gradient and controlled by mild uncoupling via proton leakage (Kowaltowski et al. 2009). These processes should be extremely important in skeletal muscle mitochondria that produce a significant amount of ATP in the mammalian body and that undergo physiological changes in surrounding tissue temperature. Therefore, in the present study, our aim was to establish the effect of experimentally induced hyperthermia (42°C) and hypothermia (25°C) on the function of isolated rat skeletal muscle mitochondria. Particularly, respiratory rate activities under phosphorylating and non-phosphorylating conditions, the yield of ATP synthesis, and for the first time, mitochondrial membrane potential (∆Ψm), mitochondrial uncoupling (proton leakage) and ROS production were studied in temperatures of 25°C, 35°C and 42°C. Moreover, the oxidative capacity of rat skeletal muscle mitochondria was studied for the first time under the tested temperatures by measuring maximal activities of cytochrome c oxidase (COX) and citrate synthase (CS). The novelty of the present study lies in demonstrating that increase in proton leak, resulting from increasing assay temperature could account for the reduced OXPHOS efficiency and ROS production.
Material and methods Chemicals All chemicals were the highest available grades and were purchased from Sigma-Aldrich unless otherwise mentioned.
Animals The experiments were carried out on adult 4-6-month-old male Wistar rats weighing 475530 g. The animals were bred in the animal house at the Poznan University of Medical Sciences, Poznan, Poland. They were given free access to water and pellet food and were housed under standard humidity and temperature conditions on a 12 h light/dark cycle. Experimental protocols involving animals, their surgery and care were approved by the Local 3
Ethics Committee on Animal Experimentation in Poznan, Poland and were in compliance with the guidelines of the European Community Council Directive on the protection of animals used for scientific purposes. Animals were sacrificed, and all efforts were made to minimize suffering.
Tissue preparation and mitochondria isolation The gastrocnemius, soleus, and quadriceps muscles were quickly excised and placed into an ice-cold isolation medium that contained 100 mM sucrose, 100 mM KCl, 50 mM TrisHCl, 1 mM KH2PO4, 0.5 mM EDTA, 0.1 mM EGTA and 0.2% bovine serum albumin (BSA), pH 7.2. All subsequent steps were performed at 4°C. The excised muscles were trimmed to remove adipose and connective tissue, finely minced and homogenized. First, muscle portions were homogenized using a Polytron homogenizer (T18 basic, IKA) (three times for 2 s, at 80% power) and then by four passes with a glass-Teflon homogenizer. The homogenate was filtered through a double layer of cheesecloth and centrifuged at 700 g for 10 min. The resultant supernatant was filtered again and then centrifuged at 9,000 g for 10 min to pellet mitochondria. The mitochondrial pellet was then resuspended in the isolation medium without BSA and centrifuged at 700 g for 10 min. The supernatant was filtered and centrifuged at 9,000 g for 10 min. The final mitochondrial pellet was then suspended in a small volume (11.5 ml) of a suspension buffer containing 225 mM mannitol, 75 mM sucrose, 0.1 mM EDTA and 10 mM Tris-HCl, pH 7.2. Mitochondrial protein concentration was determined by the by the Bradford method with BSA as a standard (Bradford 1976).
Maximal citrate synthase (CS) and cytochrome c oxidase (COX) activities The maximal CS and COX activities were assayed at various temperatures (25°C, 35°C and 42°C) as described previously (Zoladz et al. 2014). CS activity was measured with a UV1650 Shimadzu spectrophotometer at 412 nm with 100 mM 5,5’-di-thiobis-(2-nitrobenzoic acid) (TNB) and 40-60 µg of muscle mitochondrial protein. CS activity was expressed as nmol TNB x min-1 x mg protein-1. COX activity was measured polarographically using a Clark-type oxygen electrode (Hansatech) in 1 ml of a respiration medium containing 225 mM mannitol, 75 mM sucrose, 10 mM KCl, 5 mM KH2PO4, 0.5 mM EDTA, 0.5 mM EGTA, 0.05% BSA, 10 mM Tris-HCl, pH 7.2, with 0.1-0.12 mg of muscle mitochondrial protein. COX activity was assessed in the presence of sequentially added antimycin A (10 ȝM), 8 mM ascorbate, 0.06% cytochrome c, and up to 1 mM (N,N,N’N’-tetramethyl-p-phenylenediamine (TMPD). COX activity was expressed in nmol O x min-1 x mg protein-1. 4
Measurements of mitochondrial respiration and membrane potential Measurements were performed at various temperatures (25°C, 35°C and 42°C). Oxygen uptake was determined polarographically using a Rank Bros. oxygen electrode or a Hansatech oxygen electrode in either 2.8 ml or 0.7 ml of the respiration medium (see above), with either 1 or 0.25 mg of mitochondrial protein (0.36 mg/ml). The temperature of the electrode chambers was tightly controlled with Lauda Ecoline 003 thermostats. Oxygen electrodes were calibrated after each change in assay temperature. ǻȌm was measured simultaneously with oxygen uptake using a tetraphenyl-phosphonium (TPP+)-specific electrode. The TPP+electrode was calibrated after each change in assay temperature by four sequential additions (0.4, 0.4, 0.8, and 1.6 µM) of TPP+. After each run, 0.5 µM carbonyl cyanide ptrifluoromethoxyphenyl-hydrazone (FCCP) was added to release TPP+ for base-line correction. For the calculation of the ǻȌ value, the matrix volume of skeletal muscle mitochondria was assumed to be 2.0 ȝl × mg−1 protein. The values of ∆Ψm were corrected for TPP+ binding using the apparent external and internal partition coefficients of TPP+ (WoydaPloszczyca & Jarmuszkiewicz 2011). The correction shifted the calculated ∆Ψm values to lower values (approx. 30 mV-shift), but did not influence the changes in the resulting membrane potential (relative changes). The values of ǻȌm were given in mV. Succinate (5 mM) with rotenone (2 µM) or malate (5 mM) plus pyruvate (5 mM) were used as respiratory substrates. OXPHOS studies were performed in the absence of Mg2+ to avoid the adenine nucleotide interconversion catalyzed by mitochondrial adenylate kinase. Phosphorylating respiration (state 3) was measured after an ADP pre-pulse (50 µM) using 150 µM ADP as a main pulse. The total amount of oxygen consumed during state 3 respiration was used for calculation of the ADP/O ratio. Measurements of ∆Ψm allowed for fine control of the duration of state 3 respiration. The proton-conductance response to a driving force can be expressed as the relationship between the oxygen consumption rate and the ∆Ψm (flux/force relationship) when varying the potential by titrating with respiratory-chain inhibitors. Proton leak assessments during nonphosphorylating (resting, state 4) respiration were performed as previously described (SwidaBarteczka et al. 2009) with 5 mM succinate (plus 2 µM rotenone) as an oxidizable substrate in the absence of exogenous ADP and the presence of 1.8 ȝM carboxyatractyloside and 0.7 µg/ml (2 µg per mg of protein) oligomycin, which inhibited the activities of an ATP/ADP antiporter and ATP synthase, respectively. MgCl2 (0.5 mM) was added to the respiration
5
medium (see above). To induce uncoupling protein (UCP2/3) activity, linoleic acid (up to 16 µM) was used. To inhibit UCP2/3 activity, 2 mM GTP was applied. To decrease the rate of the Q-reducing pathway, succinate dehydrogenase was titrated with malonate (up to 1.7 mM).
Assay of H2O2 production by isolated mitochondria Mitochondrial H2O2 production was measured by the Amplex Red-horseradish peroxidase method (Invitrogen) (Zhou et al. 1997). Horseradish peroxidase (0.1 units/ml) catalyzes the H2O2-dependent oxidation of nonfluorescent Amplex Red (5 µM) to fluorescent resorufin red. Fluorescence was kinetically followed for 15 min at an excitation wavelength of 545 nm and an emission wavelength of 590 nm using a Infinite M200 PRO Tecan multimode reader that was adjusted internally to different temperatures. Mitochondria (0.08-0.1 mg of mitochondrial protein) were incubated in 0.5 ml of the respiration medium (see above) with 5 mM succinate as an oxidizable substrate in the absence or presence of rotenone (2 µM), oligomycin (0.7 µg/ml), carboxyatractyloside (1.8 µM) or 0.5 mM MgCl2. Reactions were monitored with constant stirring and calibrated with known amounts of H2O2. H2O2 production rates were determined from slopes calculated from readings obtained along several 15-min repeated measurements.
The determination of quinone (Q) reduction level The reduced Q versus the total endogenous pool of Q in the inner mitochondrial membrane was determined by an extraction technique followed by HPLC measurements (Jarmuszkiewicz et al. 2004; Swida et al. 2008). For calibration of the HPLC peaks, commercial Q9 was used (Sigma-Aldrich cat. no. 27597).
Statistical analysis The results are presented as the means ± S.D. obtained from 6-13 independent mitochondrial isolations, in which each determination was performed at least in duplicate. An unpaired two-tailed Student’s t-test was used to identify significant differences; in particular, differences were considered to be statistically significant if p < 0.05 (*), p < 0.01 (**), or p < 0.001 (***).
Results
6
Mitochondrial oxidative capacities increased under hyperthermia and decreased under hypothermia The oxidative capacities were elucidated by measuring the maximal COX and CS activities at various temperatures (Fig. 1). When the temperature of the incubation medium was increased from 35ºC to 42ºC, a value to which mammalian muscle temperature can increase during exercise (Saltin et al. 1968), activities of both enzymes significantly enlarged by approximately 56% and 26% for COX and CS, respectively (Fig. 1). Conversely, under hypothermia conditions, when the assay temperature was decreased from 35ºC to 25ºC, a significant reduction by approximately 32% and 21% of COX and CS activities, respectively, was observed. Thus, in rat muscle mitochondria, the oxidative capacities of the respiratory chain and TCA cycle in parallel react to changes in external temperature with both being increased or decreased under hyperthermia or hypothermia conditions, respectively.
The increase in assay temperature augmented the oxidation and phosphorylation rates but decreased the efficiency of oxidative phosphorylation The different parameters of OXPHOS were studied as a function of temperature (Fig. 2, Table 1). For both applied respiratory substrates (succinate+rotenone and malate+pyruvate), the considerable increase in the oxidation rate with the increasing temperature was observed both in state 3 and in state 4 (Fig. 2A, Table 1). Namely, the increase in measurement temperature from 25°C to 42°C resulted in an approximately five-fold increase in the rate of non-phosphorylating respiration and an approximately three-fold increase in phosphorylating respiration. Figure 2B shows that, following oxidation rate, ATP synthesis is also elevated significantly (approximately two-fold) for both applied substrates. In contrast to the oxidation and phosphorylation rates, the OXPHOS efficiency (ADP/O ratio) and the respiratory control ratio (RCR) decreased significantly with the increasing temperature (Fig. 2C, D, Table 1), indicating a possible uncoupling. For succinate+rotenone, between 25°C and 42°C, the ADP/O ratio reduced from approximately 1.4 to 1.05 and the RCR reduced from approximately 5 to 3. For malate+pyruvate, the ADP/O ratio reduced from approximately 2.4 to 1.8, and the RCR reduced from approximately 6 to 3.7 with a temperature increase from 25°C to 42°C. Thus, although the oxidation and phosphorylation activities increased with temperature, the highest apparent ADP/O ratio and RCR were observed at 25°C, far below the physiological value.
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The ∆Ψm value, which is maximal under non-phosphorylating conditions and decreases under ADP phosphorylation, reflects the substrate oxidation (proton electrochemical gradient producing reactions), and ATP turnover and proton leak (proton electrochemical gradient consuming reactions). With an increasing temperature, no temperature-dependent statistically significant changes in ∆Ψm values were observed for phosphorylating mitochondria for both studied respiratory substrates (Fig. 2E, Table 1). However, the increase in assay temperature from 25°C to 42°C resulted in a significant decrease in ∆Ψm in state 4; for succinate+rotenone, the decrease was from approximately 174 mV to 167 mV and for malate+pyruvate, the decrease was from approximately 162 mV to 156 mV. These changes were accompanied by a significant decrease in depolarization (change in ∆Ψm in response to ADP phosphorylation) from approximately 33 mV to 23 mV and from approximately 22 mV to 15 mV, for succinate+rotenone and malate+pyruvate, respectively. Thus, in rat skeletal muscle mitochondria, the lower ∆Ψm values of non-phosphorylating respiration and the decreased depolarization during the state 4 to state 3 transition, strongly suggest that the lower ADP/O and RCR ratios are the consequence of mitochondrial uncoupling increasing together with surrounding temperature.
The ATP turnover-independent proton leakage increased with the increasing assay temperature A possible modification of proton leakage was suggested by the OXPHOS studies discussed above. The proton electrochemical gradient (with ∆Ψm as the main component) is produced by reducing substrate oxidation and consumed by ATP synthesis (ADP phosphorylation) and ATP turnover-independent proton leakage. Proton leakage decreases the OXPHOS efficiency. Figure 3 shows proton conductance kinetics (the relationships between oxygen uptake and ∆Ψm obtained during progressive inhibition of the respiratory chain) in the absence or presence of OXPHOS inhibitors (oligomycin and carboxyatractyloside) for the three tested temperatures. In the absence of added ADP and OXPHOS inhibitors, respiration involves proton leak-driven respiration and endogenous ATP turnover-driven respiration. In the presence of OXPHOS inhibitors, proton leak kinetics can be analyzed. The non-ohmic curves for proton conductance were obtained for temperatures of 25°C, 35°C and 42°C. For all temperatures, addition of OXPHOS inhibitors shifted the proton conductance kinetic curves to the higher ∆Ψm and lower respiratory rate values, indicating the exclusion of ATP turnover-linked conductance. Namely, approximately 6 mV, 7 mV and 9 mV of OXPHOS
8
inhibitor-induced increases in ∆Ψm values were observed for initial, uninhibited respiratory rates for 25°C, 35°C and 45°C, respectively (Fig. 3A, B). With increasing assay temperature, the relationships between oxidation rate and ∆Ψm were shifted to the lower ∆Ψm and higher respiratory rate values, both in the absence or presence of OXPHOS inhibitors (Fig. 3A, B). Moreover, at a higher measurement temperature, a steeper relationship between oxidation rate and ∆Ψm was observed. Figure 3C presents respiratory rates measured at common ∆Ψm (157 mV) for all tested temperatures in the absence (proton leak-driven respiration and ATP turnover-driven respiration) or presence (proton leak-driven respiration) of OXPHOS inhibitors. The results indicate that the ATP turnover-independent proton leakage, together with the ATP turnover-linked proton conductance, increased with increasing assay temperature from 25°C to 42°C in rat skeletal muscle mitochondria.
The increase in assay temperature increased UCP-mediated proton leakage To exclude inducible fatty acid-mediated leak through ATP/ADP antiporter, all measurements of UCP-mediated uncoupling were performed in the presence of carboxyatractiloside. Unlike the ATP/ADP antiporter-mediated proton conductance, which can be inhibited by GDP, UCPs are strongly inhibited by GTP, in addition to GDP (WoydaPloszczyca & Jarmuszkiewicz 2014). Because, the inhibition of mitochondrial proton conductance by GTP but not GDP should be considered diagnostic of UCP function, we used GTP to determine UCP activity in isolated rat skeletal muscle mitochondria (Fig. 4 , 6). To investigate UCP-mediated proton conductance, the flux-force relationships were obtained in the presence of a low concentration (16 µM) of linoleic acid and 2 mM GTP in non-phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C (Fig. 4). In general, kinetic curves of linoleic acid-induced GTP-inhibited proton leak (UCP activity) were shifted to the lower ∆Ψm and higher respiratory rate values with increasing measurement temperature (Fig. 4A). At a higher temperature, linoleic acid induced a more pronounced mitochondrial uncoupling (a stronger increase in oxygen consumption and a stronger decrease in ∆Ψm), while GTP caused a greater inhibition of this uncoupling (a stronger decrease in the oxidation rate and a stronger increase in ∆Ψm). At the highest common ∆Ψm value (162 mV), between 25°C and 45°C, the linoleic acid-induced proton leak and the linoleic acid-induced GTP-inhibited proton leak increased approximately four
9
times (Fig. 4B). Thus, we can conclude that in rat skeletal muscle mitochondria, UCPmediated uncoupling elevated with increasing assay temperature.
At a given temperature, mitochondrial ROS production lowered under the phosphorylating and uncoupling conditions Figure 5A shows a comparison of H2O2 production of isolated rat skeletal muscle mitochondria under non-phosphorylating and phosphorylating conditions. Because with malate-pyruvate (complex I substrates), an approximately 2.4 times lower H2O2 production rate was found (at a given temperature) compared to succinate (complex II substrate), results for phosphorylating mitochondria are presented only with succinate. At all tested assay temperatures (25°C, 35°C and 42°C), rates of H2O2 production during succinate oxidation were elevated approximately 2.5-4.5 times in resting state 4 compared to phosphorylating state 3. Mitochondrial ROS production was higher under state 4 conditions, because ∆Ψm across the inner mitochondrial membrane (Fig. 2E) and the Q reduction level (Fig. 5B) were high. At all tested assay temperatures, H2O2 production by succinate-oxidizing nonphosphorylating mitochondria was approximately 32-37% lower in the presence of rotenone compared to conditions without the inhibitor of complex I (Fig. 5C). Therefore, measurements of H2O2 production in phosphorylating mitochondria were performed in the absence of rotenone (Fig. 5A). Mitochondrial ROS production is thought to be a direct function of the proton electrochemical potential and to be controlled by mild uncoupling via proton leakage through the inner mitochondrial membrane (Kowaltowski et al. 2009). Therefore, at all tested assay temperatures, in the presence of MgCl2 (which inhibits proton conductance in rat skeletal muscle mitochondria) (Cadenas & Brand 2000), oligomycin and carboxyatractyloside (which inhibit the ATP turnover-linked H+ leak), a 6-8 times higher H2O2 production was observed under non-phosphorylating conditions compared to state 4 without these additions (Fig. 5C). Higher H2O2 production in the presence of MgCl2, oligomycin and carboxyatractyloside indicates the exclusion of ATP turnover-linked uncoupling (H+ leak). Moreover, at all tested assay temperatures, a low concentration of linoleic acid (10 µM) decreased H2O2 production, while the subsequent addition of GTP (2 mM) partially restored H2O2 production (Fig. 6A). The free fatty acid-induced purine nucleotide-inhibited change in H2O2 production can be attributed to the UCP-mediated uncoupling (H+ leak) that leads to a stimulation of respiration,
10
partial ∆Ψm depolarization and decrease in Q reduction level (Fig. 4) (Jarmuszkiewicz et al. 2004 for Q reduction level). Thus, we can conclude that in rat skeletal muscle mitochondria, at a given assay temperature, ROS production decreased both under phosphorylating conditions and under uncoupling conditions (with the ATP turnover-linked and UCP-mediated leakages), and thus, at lowered ∆Ψm and Q reduction level.
Under non-phosphorylating conditions, mitochondrial ROS production and Q reduction level reduced with the increasing assay temperature When analyzing the effect of temperature on ROS production in non-phosphorylating mitochondria, a significant increase in H2O2 production (and Q reduction level) at a lower temperature (25ºC) and a significant decrease in H2O2 production (and Q reduction level) at a higher temperature (42ºC) compared to control conditions (35ºC) were observed with succinate and malate+pyruvate as respiratory substrates (Fig. 5A, B). This temperature dependence was observed in non-phosphorylating (state 4) succinate-oxidizing mitochondria both in the absence and presence of rotenone, MgCl2, oligomycin, carboxyatractyloside (Fig. 5) or linoleic acid (Fig. 6) thus independent of the ATP turnover-linked and UCP-mediated leakages. However, no temperature-dependent statistically significant changes in H2O2 production and Q reduction level were observed for phosphorylating mitochondria (state 3) (Fig. 5A, B). Thus, the increase in temperature from 25ºC to 42ºC led to the reduction in ROS production only under non-phosphorylating conditions that was accompanied by a significant decrease in Q reduction level (Fig. 5B) and state 4/ state 3 H2O2 production ratio (from approximately 4.5 to 2.5) (Fig. 5A). Moreover, the linoleic acid-induced GTP-inhibited changes in H2O2 production, which can be attributed to the UCP activity, elevated with the increase in the temperature from 25ºC to 42ºC (Fig. 6B). Thus, we can conclude that under non-phosphorylating conditions independent of the ATP turnover-linked and UCP-mediated leakages, mitochondrial ROS production and Q reduction level diminished with the increasing assay temperature.
Discussion For the first time, the role of the inner membrane conductance to protons in the mitochondrial OXPHOS process and mitochondrial ROS production was studied in skeletal
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muscle mitochondria during experimentally induced hypothermia (25ºC) and hyperthermia (42ºC) compared to the physiological temperature of resting muscle (35ºC). For nonphosphorylating skeletal muscle mitochondria, increasing the temperature from 25ºC to 42ºC led to a decrease in ∆Ψm, H2O2 production and Q reduction level (Figs. 2 and 5, Table 1). For phosphorylating mitochondria, no temperature-dependent changes in these mitochondrial functions were observed. Moreover, the efficiency of OXPHOS decreased, while the oxidation and phosphorylation rates and oxidative capacities of rat skeletal muscle mitochondria increased with increasing assay temperature (Figs. 1 and 2, Table 1). We found a significant decrease (by ~20%) in the ADP/O ratio and an acceleration of respiratory rates (state 3 by ~50% and state 4 by ~100%) and phosphorylation rate (by ~20-35%) in response to an increase in temperature of mitochondria from 35°C to 42°C (Table 1, Fig. 2). With increasing assay temperature, accelerated oxidation rate, resulting from increased proton leak, leads to accelerated ADP phosphorylation rate (respiration x ADP/O ratio) despite a decrease in the efficiency of OXPHOS (ADP/O ratio). The respiratory acceleration and a pronounced decrease in ADP/O ratio with increasing assay temperature have been previously observed with rat skeletal muscle mitochondria and rat liver mitochondria exposed to various temperatures (Brooks et al. 1971; Willis & Jackman 1994; Dufour et al. 1996). Namely, in rat skeletal muscle mitochondria, an increase in measurement temperature from 37°C to 43-45°C result in a significant increase in the phosphorylating (state 3) and non-phosphorylating (state 4) respiratory rates and in a decrease in ADP/O ratio (Brooks et al. 1971, Willis and Jackman 1994). An opposite effect, i.e., a pronounced decrease in respiratory rates has been observed during decreasing temperature from 37°C to 25°C, while ADP/O ratio remains unchanged (Brooks et al. 1971). In rat liver mitochondria, although oxidation and phosphorylation rates decrease with temperature (from 37°C to 4°C), the ADP/O ratio exhibits a maximum at 25°C (Dufour et al. 1996). Similarly to our results, in mice brain mitochondria, an increase in temperature from 32°C to 37°C resulted in respiratory acceleration, which was associated with a decrease in ROS production and attenuation of mitochondrial ∆Ψm (Ali et al. 2010). In rat skeletal muscle mitochondria, we demonstrated for the first time an increase in proton leak, including UCP-mediated proton leak (Fig. 4), with increasing assay temperature that could explain the lowering of OXPHOS efficiency and ROS production. However, one must remember that the increased temperature could also induce the leakiness of the inner mitochondrial membrane through membrane phospholipids or lipid-protein interactions (a basal proton conductance). The temperature increases could impact the integrity of the membrane, the embedded membrane proteins, the diffusion of molecules across the membrane and the activities
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of enzymes. In contrast to skeletal muscle mitochondria, in rat liver mitochondria, lack of
change in proton leakage (no increased uncoupling) between 25°C and 35°C does not account for the decrease in ADP/O ratio and RCR at the higher temperature (Dufour et al. 1996). Therefore, it seems that control of the mitochondrial proton leakage by temperature, which can influence the OXPHOS efficiency and ROS production level, depends on tissue. It could be at least partly related to the differences in the basal metabolic rates observed in various organs and tissues. It has been reported that the basal metabolic rate expressed per unit of mass in the liver (similar to its level in the brain) is approximately 20 times higher than in the skeletal muscle (Elia et al. 1992; Wang et al. 2012; Kummitha et al. 2014). Moreover, the results concerning the effect of hypothermia and hyperthermia on ADP/O ratio depend on the studied temperature range. In rat liver mitochondria exposed to varied temperatures in the range of 37°C to 4°C, a significant temperature dependence of ADP/O ratio has been observed with a maximum at 25°C (Dufour et al. 1996). In king penguin chick pectoralis muscle mitochondria, the ADP/O ratio was higher at 30°C than at 38°C (Monternier et al. 2014). The applied experimental procedure, i.e., the decreasing and increasing temperature of isolated mitochondria in relation to 35°C (a basal physiological temperature) is similar to cooling-down and warming-up of muscles in vivo by their exposure to the low or high temperature, respectively. In the present study, we demonstrated that an increase in temperature from 25°C to 42°C had a strong impact on maximal muscle COX and CS activities (Fig. 1A and B). It is worth noting that an elevation of temperature from 35°C to 42°C resulted in an 8% increase in COX activity and a 3.7% increase in CS activity per 1°C. An opposite effect, i.e., a decrease in the COX and CS activities (by 6.8% and by 3.2% per 1°C, respectively) was found when decreasing the temperature from 35°C to 25°C (Fig. 1A and B). This effect clearly shows that muscle mitochondrial OXPHOS capacity, as judged by the magnitude of maximal COX activity (Larsen et al. 2012; Zoladz et al. 2014), can be acutely modulated by changes in the muscle temperature. In the present study, we also determined the effect of varied temperatures on mitochondrial ROS production. We found 3-5-fold lower H2O2 production in state 3 compared to state 4 (Fig. 5A), indicating that H2O2 production in phosphorylating mitochondria is several times lower than in non-phosphorylating mitochondria. This is in agreement with the earlier studies showing that a high proton motive force in state 4 is potentially dangerous for the cell due to an increase in the probability of superoxide formation (Korshunov et al. 1997). Our results suggest that mitochondrial ROS production in working 13
muscles (likely with predominant phosphorylating mitochondria) is much lower than in muscles at rest (likely with predominant non-phosphorylating mitochondria). Moreover, in the present study we found that an increase in temperature from 25°C to 42°C had no effect on mitochondrial H2O2 production in state 3 and even decreased H2O2 production in state 4 (Fig. 5A). These results suggest that exposure of working muscles to high temperatures would not enhance mitochondrial ROS production, while exposure of non-working muscles to low temperatures would increase mitochondrial ROS production. Thus, our results suggest that the changes of muscle temperature might have different effects on mitochondrial ROS production in skeletal muscle at rest when compared to working muscle conditions.
Conclusions We found that temperature had a strong impact on the functional properties of skeletal muscle mitochondria. Increasing the assay temperature within the range of 25-42°C strongly increased the mitochondrial respiratory chain (including COX) and CS activities, resulting in an elevated phosphorylation rate. Moreover, temperature-induced decreases in OXPHOS efficiency and mitochondrial ROS production were observed, which may result from the temperature-induced increase in proton leak, including UCP-mediated proton leak. In skeletal muscle mitochondria, an increase in temperature from 25°C to 42°C decreased mitochondrial ROS production under non-phosphorylating conditions (likely predominant in resting muscle), while it had no effect on mitochondrial ROS production in phosphorylating mitochondria (likely predominant in working muscle).
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Acknowledgements This work was supported by the grant of the Polish Ministry of Science and Higher Education (Harmonia 2013/08/M/NZ7/00787). We thank Malgorzata Budzinska and Wioletta Nobik for technical assistance.
FIGURE LEGENDS
Fig. 1. Maximal citrate synthase (CS) and cytochrome c oxidase (COX) activities of rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. The data are presented as the means ± S.D. (n = 15) and are from 5 independent mitochondrial preparations (triplicate assays for each condition). ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar) . Fig. 2. Temperature dependence of various energetic parameters with succinate or malate + pyruvate as respiratory substrates. (A) Respiratory rates were measured in the absence (state 4, st. 4) or presence (state 3, st. 3) of 150 µM ADP with 5 mM succinate (plus 2 µM rotenone) or 5 mM malate plus 5 mM pyruvate. The corresponding phosphorylation rate (B) and the respiratory control (C) and ADP/O ratios (D) are presented. The ADP phosphorylation rate is equal to state 3 respiration × ADP/O. The respiratory control ratio is equal to the ratio of state 3 to state 4 respiration. Membrane potential values (E) were measured under state 4 (∆Ψm4) and state 3 (∆Ψm3) conditions. The resulting ∆Ψm depolarization (∆Ψm4 minus ∆Ψm3) is also presented (F). (A-F) Every data point represents the mean ± S.D. (n = 18) for at least nine independent mitochondrial preparations, in which every condition was performed in duplicate. ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar). Fig. 3. Mitochondrial membrane conductance to protons at various temperatures. Respiratory rates and ∆Ψm were measured simultaneously during progressive inhibition by malonate (up to 1.7 mM) of the respiratory chain oxidizing succinate (5 mM) plus rotenone (2 µM) in the absence (A) or presence (B) of 0.7 µg/ml oligomycin (olig) and 1.8 µM carboxyatractyloside (CATR). (C) Respiratory rates at 157 mV in the absence or presence of oligomycin and carboxyatractyloside. Experiments were performed on mitochondria incubated at 25°C, 35°C or 42°C. The data are presented as the means ± S.D. (n = 15) and are from 5 independent mitochondrial preparations (triplicate assays for each conditions). ***, p < 0.001 versus values obtained at 35°C. Fig. 4. Kinetics of linoleic acid-induced, GTP-inhibited proton leak (UCP activity) in nonphosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. (A) Succinate (plus rotenone) oxidation was gradually decreased by increasing the concentration of malonate (up to 1.4 mM) in the presence of 0.7 µg/ml oligomycin (olig) and 1.8 µM carboxyatractyloside (CATR), in the absence or presence of 16 µM linoleic acid (LA) and in the absence or presence of 2 mM GTP. Data from representative experiment (mitochondrial preparation) are presented as the means ± S.D. (n = 3) (triplicate assays for each condition). Vertical lines indicate the linoleic acid-induced GTP-inhibited proton leak size at the highest common ∆Ψm value (at 162 mV) for all tested temperature conditions. (B) Values of the linoleic acid-induced proton leak and the linoleic acid-induced GTP-inhibited proton leak 17
obtained at 162 mV are presented. ***, p < 0.001, **, p < 0.01, versus values obtained at 35°C.
Fig. 5. H2O2 production (A, C) and Q reduction level (B) in non-phosphorylating and phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. (A, B) Measurements were performed with 5 mM succinate (in the absence of rotenone) or 5 mM malate plus 5 mM pyruvate in the absence (state 4, st. 4) or presence (state 3, st. 3) of 1 mM ADP. (A) The state 4/state 3 H2O2 production ratio is shown. (C) Effect of rotenone (2 ȝM) and oligomycin (olig, 0.7 µg/ml), carboxyatractyloside (CATR, 1.8 µM) and MgCl2 (0.5 mM) on H2O2 production of non-phosphorylating succinate-oxidizing mitochondria. (A, C) The data presented as the means ± S.D. (n = 24) are from 8 independent mitochondrial preparations (triplicate assays for each experiment). (B) The data (± S.D., n = 9) are from 3 independent mitochondrial preparations (triplicate assays for each experiment). ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a vertical grey bar). Fig. 6. H2O2 production under linoleic acid-induced, GTP-inhibited proton leak (UCP activity) in non-phosphorylating rat skeletal muscle mitochondria at 25°C, 35°C and 42°C. H2O2 production was measured under non-phosphorylating conditions with 5 mM succinate in the presence of oligomycin (olig, 0.7 µg/ml), carboxyatractyloside (CATR, 1.8 µM) and MgCl2 (0.5 mM). Linoleic acid (LA) (10 µM) and GTP (2 mM) were used to activate or inhibit the UCP activity, respectively. (B) The linoleic acid-induced changes in H2O2 production (difference in H2O2 production measured the presence of linoleic acid and in the absence of fatty acid) and the linoleic acid-induced GTP-inhibited changes in H2O2 production (difference in H2O2 production measured the presence of linoleic acid and in the presence of linoleic acid and GTP). (A, B) The data presented as the means ± S.D. (n = 24) are from 8 independent mitochondrial preparations (triplicate assays for each experiment). **, p < 0.01, *, p < 0.05 versus values obtained at 35°C.
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Table 1. Respiratory rates, ∆Ψm values and coupling parameters in rat skeletal muscle mitochondria at 25°C, 35°C and 42°C Experimental conditions are similar to those in Fig. 2. The respiratory rates (in nmol O x min1 x mg-1 protein) and ∆Ψm values (in mV) of state 3 (phosphorylating respiration) and state 4 (non-phosphorylating respiration following state 3) as well as corresponding respiratory control ratios (RCR) and ADP/O ratios are presented. Mean values (± S.D.) for at least nine different mitochondria preparations (with duplicate measurements, n = 18) are shown. ***, p < 0.001, **, p < 0.01, *, p < 0.05 versus values obtained at 35°C (a grey-shadowed column). malate + pyruvate 25°C 35°C 42°C State 3 197 ± 22*** 421 ± 45 609 ± 105* 141.7 ± 0.7 141.4 ± 2.2 ǻΨm3 140.5 ± 1.2 State 4 32.9 ± 3.4*** 84.9 ± 5.9 164 ± 13** ǻΨm4 162.2 ± 1.5* 159.4 ± 1.3 156.1 ± 2.0* RCR 5.98 ± 0.42* 4.96 ± 0.44 3.70 ± 0.2** ADP/O 2.43 ± 0.12 2.25 ± 0.16 1.79 ± 0.24** succinate + rotenone 25°C 35°C 42°C State 3 335 ± 32*** 602 ± 88 951 ± 61*** 143.2 ± 1.2 144.1 ± 1.4 ǻΨm3 141.0 ± 0.9 State 4 65.6 ± 6.1*** 150 ± 16 302 ± 35** 174.0 ± 1.9* 171.2 ± 0.7 167.4 ± 1.3* ǻΨm4 RCR 5.11 ± 0.24** 4.02 ± 0.34 3.15 ± 0.26** ADP/O 1.39 ± 0.06* 1.23 ± 0.08 1.06 ± 0.09*
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