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• W1592611595 abstract "What is the central question of this study? The central question was to establish whether decreased cholesterol content in heart mitochondria caused by prolonged swimming may provoke changes in their bioenergetics and affect resistance to CaCl2-induced mitochondrial swelling. What is the main finding and its importance? The main finding is the indication that changes in the cholesterol pool in heart mitochondria induced by swimming exercise are related to an increase in resistance to CaCl2-induced swelling, probably by remodelling of lipid microdomains, and are not deleterious for mitochondrial bioenergetics. These findings may contribute to a more complete understanding of the defense system that may prevent mitochondrial degradation during exercise and the protective system of cardiac cell defense in stress conditions. The significance of the reduction of the cholesterol pool in heart mitochondria after exercise is still unknown. Recently, published data have suggested that cholesterol may influence the components of mitochondrial contact site and affect mitochondrial swelling. Therefore, the aim of this study was to determine whether the decreased cholesterol content in heart mitochondria caused by prolonged swimming may provoke changes in their bioenergetics and result in an increased resistance to calcium chloride-induced mitochondrial swelling. Male Wistar rats were divided into a sedentary control group and an exercise group. The rats exercised for 3 h, burdened with an additional 3% of their body weight. Their hearts were removed immediately after completing the exercise. The left ventricle was divided and used for experiments. Mitochondrial cholesterol content, membrane fluidity and mitochondrial bioenergetics were measured in the control and exercised rat heart mitochondria. To assess whether mitochondrial modifications are linked to disruption of lipid microdomains, methyl-β-cyclodextrin, a well-known lipid microdomain-disrupting agent and cholesterol chelator, was applied to the mitochondria of the control group. Cholesterol depletion, increased membrane fluidity and increased resistance to calcium chloride-induced swelling were observed in postexercise heart crude mitochondrial fraction. Similar results were achieved in control mitochondria treated with 2% methyl-β-cyclodextrin. All of the mitochondrial bioenergetics parameters were similar between the groups. Therefore, the disruption of raft-like microdomains appears to be an adaptive change in the rat heart following exercise. Acute physical exercise is related to oxidative stress. The role of oxidative stress that is caused by an overaccumulation of reactive oxygen species or reactive nitrogen species during exercise has been discussed over the past 30 years (for a review, see Powers et al. 2011a). These highly reactive molecules may damage tissues and macromolecules (Davies et al. 1982; Liu et al. 2000), but they are also necessary signalling agents in the adaptation process induced by exercise training (Powers et al. 2011b). Oxidative stress is a well-known important factor implicated in regulation of the mitochondrial permeability transition pore (mPTP; Skulachev, 2000), a multiprotein complex localized in specific mitochondrial sites, known as mitochondrial contact sites. Activation of mPTP is associated with mitochondrial depolarization, uncoupling of oxidative phosphorylation, swelling of mitochondria and release of death-promoting factors, such as cytochrome c (Bernardi et al. 1999). Intracellular calcium is another factor implicated in mPTP regulation. Physiological stimuli, such as physical exercise, that cause an increase of cytosolic free Ca2+ or the release of Ca2+ from the endoplasmic reticulum invariably induce mitochondrial Ca2+ uptake, with a rise of mitochondrial matrix free Ca2+. Hence, mitochondria accumulate Ca2+ and efficiently control the spatial and temporal shape of cellular Ca2+ signals, yet this situation exposes them to the hazards of Ca2+ overload, which can lead to opening of the mPTP. Persistent mPTP opening is followed by depolarization, with Ca2+ release, cessation of oxidative phosphorylation, matrix swelling with inner membrane remodelling and, eventually, outer membrane rupture with release of cytochrome c and other apoptogenic proteins (Bernardi & Rasola, 2007). However, mitochondria isolated from postexercise rat skeletal muscle have not demonstrated an increased susceptibility to swelling. On the contrary, these mitochondria appear to be more resistant to calcium chloride-induced swelling; therefore, it has been proposed that increased Ca2+ resistance may prevent mitochondrial degradation, although the mechanism responsible for this protection remains unknown (Fernstrom et al. 2004). Changes in the response to Ca2+-induced mitochondrial swelling may be linked to remodelling of raft-like microdomains (Ziolkowski et al. 2010). Growing evidence suggests that raft-like microdomains occur in mitochondria and are involved in signalling pathways that control the apoptotic process (Garofalo et al. 2005, 2007; Martinez-Abundis et al. 2007; Raimondo et al. 2012; Sorice et al. 2012). On the contrary, another group has reported that mitochondria do not contain lipid rafts and that lipid rafts do not contain mitochondrial proteins (Zheng et al. 2009). Rather, according to recently published data, mitochondria-associated endoplasmic reticulum membranes (MAMs) are intracellular detergent-resistant lipid raft-like domains (Area-Gomez et al. 2012; Fujimoto et al. 2012). However, in spite of the controversy related to the localization of lipid raft-like microdomains, these dynamic structures contain protein and lipid components and are associated with mitochondrial contact sites and with mPTP, and are involved in regulation of Ca2+ signalling, mitochondrial bioenergetics and apoptosis (Rizzuto et al. 1999; Voelker, 2005; Fujimoto et al. 2012; Sorice et al. 2012). It has also been shown that lipids specific to raft-like microdomains play a crucial role in mPTP regulation (Martinez-Abundis et al. 2007). Furthermore, it has been proposed that lipid microdomains that are enriched in cholesterol and ceramide may coexist as structural elements with some mPTP-forming proteins and with members of the Bcl-2 family (Martinez-Abundis et al. 2009). Cholesterol is a key element of the membrane raft-like microdomains, and changes in its amount influence their function and structure (Martinez-Abundis et al. 2007; Fujimoto et al. 2012). The cholesterol content of mitochondria is mainly distributed within areas of the outer membrane involved in creation of the mitochondrial contact sites (Ardail et al. 1990). Remodelling of mitochondrial cholesterol content has been noted in physiological conditions and in vitro. A significant reduction in the cholesterol content of cardiac mitochondria has been observed in rats treated with permethrin, an oxidative stress-inducing agent (Vadhana et al. 2011a). It has been also shown that prolonged swimming decreases the cholesterol content of cardiac mitochondria (Keatisuwan et al. 1991). Additionally, a similar decrease in cholesterol has been observed in isolated mitochondria treated with methyl-β-cyclodextrin (MβCD; Ziolkowski et al. 2010), a well-known lipid microdomain-disrupting agent and cholesterol chelator (Ohtani et al. 1989). Mitochondria treated with MβCD exhibited greater resistance to mitochondrial swelling induced by calcium chloride or recombinant BAX (rBAX) than control mitochondria (Garofalo et al. 2005; Martinez-Abundis et al. 2007; Ziolkowski et al. 2010). Thus, the changes in the mitochondrial cholesterol pool induced by exercise may influence mitochondrial contact site components and may affect mitochondrial swelling. In the present study, therefore, MβCD was used in isolated control mitochondria to test whether the changes in mitochondria observed after exercise are linked to disruption of mitochondrial lipid microdomains. We hypothesized that the decreased cholesterol content in heart mitochondria caused by prolonged swimming may provoke changes in raft-like microdomains and increase resistance to calcium chloride-induced mitochondrial swelling. Experiments were conducted in accordance with the principles of UK legislation and approved by the Local Ethics Committee of the Gdansk Medical University (consent no. 13/2007). The animals were anaesthetized by intraperitoneal injection of an overdose of ketamine and xylazine (90 and 10 mg kg−1, repectively) and, after opening the thoracic cages of the anaesthetized animals, their hearts were rapidly removed and immersed in ice-cold isolation medium (buffer A; 0.01 m Tris–HCl, 0.3 m sucrose and 0.001 m EGTA, pH 7.3) and weighed. The atrial tissue was removed and was not used in the assays. The left ventricle (except the small pieces of apical segment) and the entire right ventricle were used for preparation of heart cells and mitochondria. Male Wistar rats (n= 36, as follows: main study, n= 12; and additional study, n= 24), weighing 250–300 g, were housed in an environmentally controlled room (23 ± 1°C with a 12 h–12 h light–dark cycle) and received standard rat chow and water ad libitum. All compounds, except for bovine serum albumin (BSA; Merck, Damstadt, Germany), were purchased from Sigma (St Louis, MO, USA). The reagents 1,6-diphenyl-1,3,5-hexatriene (DPH) and 6-lauroyl-2-dimethylaminonaphthalene (Laurdan) were purchased from Molecular Probes (Eugene, OR, USA). The cholesterol assay kit was generously donated by CHEMA Diagnostica, Monsano (AN) Italy. Exercise protocol The rats were prepared for the experiments and exercise tests using the methods described by Keatisuwan et al. (1991). The rats were randomly divided into sedentary control (C) and long-lasting endurance exercise (E) groups. Before the exercise protocol, the animals in the E group were trained to reduce the stress of swimming. Each day during the preparatory procedure, the rats swam for 30 min in water at 35°C. On the first day, the rats swam without any additional weight. On the second, third and fourth days, the rats swam burdened with 1, 2 and 3% of their body weights, respectively. The weights were attached to the rats’ tails. On the fifth day, exercise testing was performed in the E group of rats; it consisted of 3 h of prolonged swimming in 35°C water burdened with 3% of their body weight. The rats were killed (as described in the ‘Animal care’ subsection) immediately after completing the swimming protocol. In additional experiments, we applied this pre-exercise procedure (P group) and pre-exercise procedure with 3 h standing in the water (S group) to control rats. We observed that a short-term preparatory swimming protocol and temperature of the water did not significantly alter cholesterol and swelling in mitochondria (additional study; Figs S1 and S2, supplementary data). Therefore, all other study parameters were assessed in C and E groups. Blood lactate analysis Immediately after the heart removal (as described in the “Animal Care” subsection) the blood samples from the thoracic cage of the anaesthetized animals were collected. Immediately after collection, the blood was deproteinized by adding ice-cold 0.4 m perchloric acid. After being thoroughly mixed, the samples were centrifuged at 12,000g for 10 min. The blood lactate levels were determined using the lactic acid oxidase method with a standard Randox Laboratories kit (kit no LC2389, Crumlin, UK), and the assays were performed on a Cecil CE9200 spectrophotometer (Cambridge, UK). Preparation of heart cells The hearts were collected, rinsed with ice-cold PBS, and the ventricles were dissected into small fragments using a surgical blade (Vadhana et al. 2010, 2011a,b). The samples were then homogenized in a glass tube with a pestle and extensively washed again in cold PBS until blood cells were not observed and then minced using 0.9% NaCl. The isolated mixed population of cells were resuspended in PBS and stored in ice. Protein content was used to normalize samples. Protein measurement The protein content was measured using the method of Lowry et al. (1951), with BSA as a standard. Isolation of rat heart mitochondria (RHM) The mitochondria were isolated as described by Marcil et al. (2006), with slight modifications. The hearts were removed, weighed and placed in an ice-cold mitochondria isolation buffer (buffer A, pH 7.3), supplemented with 0.2% BSA. The ventricular tissue was minced with scissors in 5 ml of buffer A, and subtilisin A, (1.5 mg g−1) was then added. After 5 min of incubation, the volume was made up to 30 ml with buffer A supplemented with 0.2% BSA and homogenized. The homogenate was centrifuged at 800g for 10 min. The pellet was discarded, and the supernatant was decanted, filtered through two layers of nylon gauze and centrifuged at 10,000g for 10 min. The pellet obtained was resuspended in buffer B (0.3 m sucrose, 0.01 m Tris–HCl and 0.0005 m EGTA, pH 7.3) and centrifuged at 10,000g for 10 min. After this washing step had been repeated twice, the final mitochondrial pellet was resuspended in 0.3 ml of buffer B to a protein concentration of 25 mg ml−1. All of the procedures were carried out at 4°C. Fluorescence measurements on heart cell and mitochon-drial membranes Fluorescence measurements were used to evaluate the dynamic changes in the heart cell membranes and in the freshly isolated mitochondrial membranes suspended in PBS. Two fluorescent probes, DPH and Laurdan, were used on both the heart cell and mitochondrial membranes. The final protein concentration in the assay was 0.4 mg ml−1, and the probe (DPH or Laurdan) concentration was 10−6m. A Hitachi 4500 spectrofluorometer was used for the fluorescence measurements. The steady-state DPH fluorescence anisotropy (r) was calculated using excitation and emission wavelengths of 360 and 430 nm, respectively, according to the equation of Shinitzky & Barenholz (1978). The generalized polarization of Laurdan (GP340) was calculated according to the Parasassi equation (Parasassi et al. 1990, 1991). Cholesterol estimation The cholesterol content was measured in samples that had been normalized to the mitochondrial protein concentration. The total lipids from freshly isolated heart mitochondria were prepared by vortexing with four volumes of 2:1 (v/v) methanol/chloroform. The mixture was then centrifuged at 4000g for 5 min. A 50 mm citric acid:water:chloroform solution (1:2:1 v/v/v) was added to the supernatant, and the mixture was vortexed and centrifuged at 4000g for 20 min. A lower chloroform phase, an upper water/methanol phase and an interphase that consisted mainly of precipitated protein were obtained. The upper liquid (water/methanol) phase was discarded, leaving the protein layer untouched. The lower (chloroform) phase (without any droplets of the upper phase) was transferred into a new tube. Glass or high-quality plastic pipettes were used throughout. Before transferring the chloroform phase, the pipette was prerinsed with chloroform twice. This chloroform phase now contained all of the non-polar lipids (cholesterol and its esters, mono-, di- and tri-glycerides, waxes etc.). The solvent was completely evaporated by means of vacuum drying, and the pellet was later dissolved in 20 μl of ethanol. All of the lipids were extracted from the heart mitochondria, and the total cholesterol level (in milligrams per decilitre) was determined using an enzymatic assay. In the presence of oxygen, free cholesterol is oxidized by cholesterol oxidase to cholesten-4-ene-3-one and hydrogen peroxide. The hydrogen peroxide reacts with p-chlorophenol and 4-aminoantipyrine in the presence of peroxidase to form a quinoneimine dye. The intensity of colour formed after 5 min of incubation at 37°C is proportional to the cholesterol concentration and can be measured photometrically at 510 nm. A standard cholesterol solution was used as a reference. Mitochondrial respiratory activity assay The mitochon-drial oxygen uptake was measured at 25°C with a Clark-type oxygen electrode (Oxygraph; Hansatech Instruments, Kings Lynn, UK). The respiration medium contained 170 mm sucrose, 15 mm KCl, 5 mm potassium phosphate, 0.1 mm EDTA and 0.1% BSA. Five millimolar glutamate and 5 mm malate (pH 7.4) or 5 mm succinate and 1 μm rotenone were used as oxidizable substrates. The respiratory control index and adenosine diphosphate to oxygen ratio were measured and calculated from oxygen electrode traces, as has been previously described (Estabrook, 1967). Measurement of mitochondrial enzyme activities The cytochrome c oxidase activity was measured spectrophotometrically (Cecil CE9200, Cambridge, UK) at 30°C in the mitochondrial fraction (Wharton & Tzagoloff, 1967). The enzyme activity was expressed as nanomoles per minute per milligram of protein. Swelling of the RHM The RHM swelling measurement was performed spectrophotometrically (Cecil CE9200) by the convenient swelling assay. Mitochondria (1 mg ml−1) were incubated in 1 ml of buffer C (250 mm sucrose, 10 mm MOPS and 0.05 mm EGTA, pH 7.2) containing succinate (5 mm) with rotenone (1 μm; Marcil et al. 2006). Calcium chloride (100 μm) was used as an mPTP opening inducer and ciclosporin (CSA; 1 μm) as an mPTP opening inhibitor. The swelling curves were recorded at 540 nm. Cuvettes containing the mitochondrial suspension were thermostated at 25°C. Oxidative stress parameters in rat heart homogenates Carbonyl groups (Levine et al. 1990) and lipid dienes (oxidation index 233/215; Misik et al. 1992) were measured in cardiac homogenates of C, E, P and S groups of rats. The values of carbonyl groups and lipid dienes are expressed as a percentage of the relevant control values (100%). All data are presented as means ± SEM of at least six independent experiments. Cholesterol estimation For the in vitro study, cholesterol was depleted from isolated RHM taken from the control group using MβCD. The aim of the study was to find the concentration of MβCD that led to a decrease in the mitochondrial cholesterol level similar to that observed after the exercise protocol used in the in vivo experiment. Isolated RHM (1 mg protein ml−1) were incubated with the control buffer C or with buffer C containing 1, 2, 3 or 4% MβCD. After 5 min of incubation, the samples were centrifuged at 10,000g for 10 min, and the mitochondrial cholesterol levels were determined in the pellets using the method described for the in vivo study. Swelling of the RHM The RHM swelling measurement was performed spectrophotometrically using the convenient assay described for the in vivo study. The swelling measurement was performed for the control RHM and for the 2% MβCD-treated RHM. Statistical analysis was carried out using a software package (Statistica version 10.0; StatSoft Inc., Tulsa, OK, USA). The results are expressed as means ± SEM. The differences between means were tested using Student's unpaired t test (the results of C versus E group) or a one-way ANOVA model (the results of C, E, P and S groups or C, E and 2% MβCD groups). If a difference was detected in the ANOVA model, the significant differences were determined using the Newman–Keuls post hoc test. Statistical significance was established at P < 0.05. To determine the intensity of swimming exercise, the blood lactate concentration was measured and significantly higher values were found in the exercised rats than in the control animals (3.41 ± 0.46 and 1.98 ± 0.21 mmol l−1, respectively; Fig. 1). Blood lactate concentration in control and exercised animals Blood lactate concentration was measured in control (C) and exercise groups (E). *P < 0.05 significantly different compared with control. All data are shown as means + SEM and are expressed as millimoles per litre (n= 6 in each group). To verify that exercise was able to induce oxidative stress, protein carbonyl groups and lipid dienes were measured in cardiac homogenates of C, E, P and S groups of rats. Significantly higher oxidative stress parameters were seen in only the E group compared with the C, P and S groups (Table 1 and Table S3 in supplementary material). Prolonged swimming caused a significant drop in the cholesterol content of the heart mitochondria (Fig. 2A). The mitochondria isolated from the hearts of the exercised rats had almost 67% of the cholesterol of the control rats. To verify whether the reduction of mitochondrial cholesterol is related to biogenesis of new mitochondria, the results of cholesterol content were normalized to milligrams of mitochondria (Fig. S3 and Table S2, supplemental data). The results were similar to those calculated per milligram of mitochondrial protein. We also estimated the influence of different concentrations of MβCD on the cholesterol status of rat heart mitochondria. Mitochondrial cholesterol content after MβCD at concentrations of 1, 2, 3 and 4% was 88 ± 8.5, 68 ± 4.0, 55 ± 6.5 and 49 ± 2.5%. respectively, compared with the control values (100%). As shown in Fig. 2, the decrease in RHM cholesterol content after exercise was similar to the effect of 2% MβCD; therefore, this concentration was used in the swelling study (see ‘Swelling of mitochondria’ below). Changes in the mitochondrial cholesterol content A, the total cardiac mitochondrial cholesterol levels (in micrograms per milligram of protein) for the control rats and the rats undergoing prolonged endurance exercise. The data are shown as means + SEM; *P < 0.005 versus the control rats (n= 6 in each group). B, methyl-β-cyclodextrin (MβCD)-induced cholesterol depletion in rat heart mitochondria (RHM). Methyl-β-cyclodextrin is an agent that mimics the effect of exercise. The RHM were treated with 0, 1, 2, 3 or 4% MβCD for 5 min. After centrifugation (10,000g for 5 min), the cholesterol content of the mitochondrial pellets was evaluated. All data are presented as means ± SEM of at least four independent experiments. The cholesterol content of each probe is expressed as a percentage of the relevant control level without MβCD (100%). Given that changes in cholesterol content alter the physicochemical properties of the bilayer, membrane fluidity was studied using two fluorescent probes that localize at different membrane depths. Laurdan was employed because it is present in the same region of cholesterol and provides information about the hydrophilic–hydrophobic section of the bilayer, while DPH detects the state of the hydrophobic inner part of the membrane and the physical state of fatty acids (Shinitzky & Barenholz, 1978; Parasassi et al. 1990, 1991). The generalized Laurdan GP340 polarization was significantly lower in the heart mitochondrial membranes of the rats subjected to swimming exercise for 3 h (Fig. 3A) than in the control animals. These results indicate that the hydrophilic–hydrophobic section of the bilayer, where the cholesterol levels decreased, is more fluid. Likewise, the steady-state DPH fluorescence anisotropy (Fig. 3B) decreased significantly in the rat cardiac mitochondrial membranes after exercise. This outcome indicates that more fluid fatty acids are located in the deeper hydrophobic region tested by the probe. Prolonged endurance exercise significantly changes the fluidity of mitochondrial membranes A, the generalized fluorescence polarization (GP340) of Laurdan measured in the cardiac mitochondrial membranes of the control rats and the rats undergoing prolonged endurance exercise. B, the steady-state DPH fluorescence anisotropy (r) in the cardiac mitochondrial membranes for the control rats and the rats undergoing prolonged endurance exercise. The samples were normalized to a 0.4 mg ml−1 protein concentration in PBS. The data are shown as the means + SEM; *P < 0.05 versus the control rats (n= 6 in each group). In order to evaluate whether exercise can modify the fluidity of the plasma membrane, fluorescent probes were also employed in the heart cells. The exercised group showed significantly lower GP340 values than the control group (Fig. 4A), implying that the membrane is more fluid in the hydrophilic–hydrophobic region of the bilayer that is tested by Laurdan. Furthermore, the steady-state DPH fluorescence anisotropy showed a significant increase, corresponding to a less fluid membrane in the hydrophobic part of the lipid bilayer in heart cells from the exercised group (Fig. 4B). Prolonged endurance exercise significantly changes the fluidity of heart cell membranes A, generalized fluorescence polarization (GP340) of Laurdan measured in the heart cell membranes between control rats and rats that underwent prolonged endurance exercise. B, steady-state fluorescence anisotropy (r) of DPH in the heart cell membranes between control rats and rats that underwent prolonged endurance exercise. Data are shown as the means + SEM; *P < 0.05 versus control rats (n= 6 in each group). In order to verify whether depletion of cholesterol from cardiac mitochondria changes its bioenergetics, mitochondrial respiratory and cytochrome c oxidase activity were determined. All of the mitochondrial bioenergetics parameters were similar in both groups (Table S1, supplemental data). Finally, we determined whether mitochondrial cholesterol depletion was associated with mitochondrial swelling. The calcium chloride-induced mitochondrial swelling was significantly less in the rat cardiac mitochondria isolated after exercise and in the control mitochondria treated with 2% MβCD than in the control group (Fig. 5A and B). The inhibitory effects of 1 μm CSA (added at the beginning of incubation) were observed (Fig. 5A). Elevated absorbance was also detected after adding calcium chloride in the presence of CSA, indicating mitochondrial shrinkage (Fig. 5A). Similar effects of exercise and MβCD on calcium chloride-induced mitochondrial swelling A, prolonged endurance exercise decreased calcium chloride-induced RHM swelling. The calcium chloride-induced mitochondrial swelling was assessed spectrophotometrically. An inhibitory effect was observed for 1 μm ciclosporin (CSA; added at the beginning of incubation). Exercise and 2% MβCD (a cholesterol chelator) partly reduced the CaCl2-induced swelling. The arrow indicates the time when the CaCl2 was added. Typical curves for at least six independent experiments are presented. B, the mean values of the absorbance changes measured at 540 nm (swelling of mitochondria). *P < 0.01, significantly different from the control group (n= 6 in each group). In the present study, we demonstrate that adaptive responses to exercise are related to an increase in isolated mitochondrial resistance to Ca2+-induced swelling. In support of our hypothesis, we observed that exercise induced a decrease in mitochondrial cholesterol content. Similar effects obtained using MβCD suggested that the mechanism responsible for this phenomenon might be linked to remodelling of the raft-like microdomains. These results are in agreement with previously published data (Keatisuwan et al. 1991; Perez et al. 2003). Simultaneously, no changes in the bioenergetic status of mitochondria of exercise rats were noted. This finding implies that changes in cholesterol level, mitochondrial fluidity and mitochondrial sensitivity to swelling are not deleterious. Rather, these changes indicate a dynamic physiological process that may help mitochondria adapt to stress conditions. The varied response of mitochondria associated with changes in the cholesterol pool during stress circumstances has been observed. As has been recently reported, the mitochondrial cholesterol content of rat hearts is reduced during oxidative stress (Vadhana et al. 2011a). Lower cholesterol levels in these mitochondria have been associated with DNA damage, increased protein carbonyl content and lipid hydroperoxide formation in cardiac cells. Also in our study, elevated parameters of oxidative stress in postexercise hearts were observed in comparison to the control animals. On the contrary, significant accumulation of mitochondrial cholesterol has been documented in ischaemic myocardial processes (Rouslin et al. 1980; Rouslin et al. 1982). Mitochondria from the ischaemic area after 2 h of occlusion of the left anterior descending coronary artery exhibited an 89% increase in cholesterol content with no change in either total phospholipid content or membrane fatty acids composition (Rouslin et al. 1982). Moreover, increased mitochondrial cholesterol content has been proposed as a marker of mitochondrial membrane injury (Rouslin et al. 1980). The mechanism responsible for removal of cholesterol from mitochondrial membranes remains unknown, but involvement of 18 kDa mitochondrial translocator protein (TSPO) in this mechanism must be taken into consideration. This protein is responsible for the uptake of cholesterol into mitochondria of steroidogenic organs, but also in heart (Paradis et al. 2013). Also, as an element of the mitochondrial outer membrane, cholesterol is associated with the outer/inner mitochondrial membrane contact sites (Rone et al. 2009). Administration of the selective TSPO ligands inhibited oxidative stress, improved mitochondrial function, and abolished both mitochondrial cholesterol accumulation and oxysterol production induced by ischaemia–reperfusion. These data showed that reduction of cholesterol accumulation in heart mitochondria by modulation of TSPO activity prevents mitochondrial injury (Paradis et al. 2013). To the best of our knowledge, the effect of exercise on the action of TSPO in the heart has not been shown. However, changes in tissue density of TSPO have been presented following acute swim stress in the rat hippocampus, adrenal and kidney (Avital et al. 2001). To avoid the effect of psychological stress accompanying swimming exercise, all of the rats in our experiment were subjected to the swimming preparatory procedure prior to the exercise test. Nonetheless, we cannot exclude the possibility that exercise, via adaptive response to psycho-physiological stress, influences the density of TSPO in heart cells and prevents mitochondrial injury. The beneficial effects of acute exercise and long- or short-term endurance training on cardiac mPTP opening and mitochondrial swelling in different pathological conditions, such as doxorubicin treatment, ischaemia–reperfusion, hyperglycaemia or hypercholesterolaemia, have also been reported (Ciminelli et al. 2006; Ascensao et al. 2011; McCommis et al. 2011). In these conditions, the exercise training induces a stabilization of cyclophilin D (Lumini-Oliveira et al. 2011; McCommis et al. 2011), an important mPTP component and sensitizer, which correlates with the susceptibility of isolated mitochondria to undergo mPTP opening (Csukly et al. 2006; Matas et al. 2009). The interaction between cyclophilin D and cholesterol is still unknown and is a phenomenon worthy of investigation. The novelty of our study is the indication that a reduction in the cholesterol pool may be another agent associated with the defense of mitochondria against swelling during exercise stress. In order to understand the physiological role of the changes in the mitochondrial cholesterol pool after exercise, we treated the control mitochondria with MβCD. According to previously published data, MβCD decreases cholesterol levels and increases resistance to calcium induced-swelling in a dose-dependent manner (Ziolkowski et al. 2010). We found that using a 2% solution of MβCD led to decrease of cholesterol levels similar to those of the exercise hearts without causing any significant changes in bioenergetics. The Laurdan data supported this finding. This probe has been employed to evaluate the order of lipid microdomains, because it is sensitive to phase variation in ordered and fluid membrane regions and to the water and cholesterol content of the membrane (Harder et al. 2007). The correlation between cholesterol content and the GP value of Laurdan was reported by Wheeler & Tyler (2011), who showed that increasing MβCD concentrations reduced GP values in cell lines, as we found in our mitochondrial membranes. Our study found that exercise induced physicochemical changes in mitochondrial membranes and in cardiac cells. Skilled domains in plasma membranes form platforms that affect protein functions in the bilayer. The physical properties of plasma membrane domains can have functional implications for receptor and ion channel activities, signalling, recognition events (Marguet et al. 2006) and for mitochondrial respiration and oxidative phosphorylation. The increased fluidity measured in both regions of the mitochondrial membrane could be associated with a remodelling process of membrane lipids that could be associated with a preparatory process aimed to enhance the formation of energy transition pores in the mitochondrial membrane as a compensatory response to the increased energy demands of the heart cells. The more fluid mitochondrial membrane could represent an endogenous mechanism of response to exercise. In contrast, an increased membrane fluidity in the hydrophilic–hydrophobic region of the bilayer and a more viscous state in the inner region were measured in the heart cells following exercise. Conformational changes of the plasma membrane can influence sodium and calcium flux, because the lipid composition of the bilayer differs between the inner and the outer leaflets, and the composition is heterogeneous in both parts. Different packing of lipids and proteins (i.e. ion channels) leads to the formation of microdomains or lipid rafts that are mobile platforms, rich in sphingolipids, cholesterol and specific proteins, actively involved in cellular and transmembrane signalling. The physicochemical properties of the plasma membrane have a crucial role in regulation of the protein activities inside the membrane. If the channel is located in a microdomain where lipids are in a fluid state, the protein, being dynamic, could work more easily then in a sticky environment. This effect depends on the lipid–lipid and lipid–protein interactions in the bilayer that is correlated not only to the type of lipids in the membrane but also to their oxidative status. The increase of carbonyl groups and dienes measured in the animals after exercise can be responsible for a decrease of bilayer fluidity in the inner region of heart cells, because the oxidized molecules establish more bonds, reducing the mobility of lipids into the bilayer and then also the dynamicity of proteins. These outcomes should be considered in future studies, because these changes may influence the dynamic properties of the membrane and the cellular exchanges influencing the sodium and calcium influx in cardiomyocytes. All together, these results suggest that exercise in physiological conditions may induce reversible remodelling of raft-like microdomains and in this way inhibit mitochondrial swelling. There are no direct lines of evidence that exercise affects the mitochondrial domains in mitochondrial membranes or in MAMs, but the inhibitory effects of both exercise and MβCD on swelling are visible. This supports previously published data reporting that depletion of mitochondrial cholesterol by MβCD results in higher resistance to mitochondrial swelling (Garofalo et al. 2005; Martinez-Abundis et al. 2007; Ziolkowski et al. 2010). Owing to the controversy about their localization, as mentioned in the Introduction, in this study we used crude mitochondria, which have both MAMs and/or mitochondrial raft-like microdomains (Wieckowski et al. 2009; Fujimoto et al. 2012). Thus, we cannot exclude the possibility that exercise affects raft-like microdomains: 1) localized near mitochondrial contact sites or 2) in MAMs, which can be involved in stabilization of Ca2+ homeostasis or 3) induces interaction between both rafts. In conclusion, in response to cell stress, postexercise mitochondria lost a considerable amount of cholesterol, exhibited increased mitochondrial membrane fluidity and showed increased resistance to calcium chloride-induced mitochondrial swelling. No significant decreases in bioenergetics were observed. These findings may contribute to a more complete understanding of the defense system that may prevent mitochondrial degradation during exercise and of the protective system of cardiac cell defense in stress conditions. None declared. The presented work was supported by the grant from the Polish Ministry of Science and Higher Education (no. N N404 168434) in the period 2008–2011. We acknowledge Chema Diagnostica, Monsano Italy for providing the kit for cholesterol assay. The short-term preparatory swimming protocol and temperature of the water did not significantly alter cholesterol (Fig. S1) and swelling in mitochondria (Fig. S2). Figure S1. The total cardiac mitochondrial cholesterol levels for four groups of rats: control (C), exercise (E), subjected short-term preparatory swimming protocol (P) and subjected short-term preparatory swimming protocol and 3 h standing in the water (S). Cholesterol levels are expressed as percentage of the relevant control values (100%). All data are presented as means ± SE of at least six independent experiments. *P < 0.005, significantly different from the C, P and S groups (n = 6 in each group). Figure S2. Prolonged endurance exercise decreased calcium chloride-induced RHM swelling, but not pre-exercise preparatory protocol or water temperature. The calcium chloride-induced mitochondrial swelling was assessed spectrophotometrically. An inhibitory effect was observed for 1 μm CSA (added at the beginning of incubation)(data not shown). The mean values of the absorbance changes measured at 540 nm are expressed as percentage of the relevant control values in 20 min (decrease in absorbance = swelling of mitochondria). *P < 0.01, significantly different from the C, P and S groups (n = 6 in each group). Figure S3. Changes in the mitochondrial cholesterol content normalized to mitochondrial content. The total cardiac mitochondrial cholesterol levels (μg (mg of mitochondria)−1) for the control rats and the rats undergoing prolonged endurance exercise. The data represent mean ± SE. *P < 0.01 versus the control rats (n = 5 in each group). Table S1. The effect of prolonged endurance exercise on mitochondrial bioenergetics. The mitochondrial bioenergetics parameters (i.e., the rate of oxygen consumption in state 3 and state 4, RCI and ADP/O) were not significantly different between the two groups (n = 6 in each group). Similarly, the cytochrome c oxidase activity was not changed after the swimming. Table S2. Mitochondrial density, Citrate Synthase activity in cardiac homogenates and mitochondria of C, E, P and S groups of rats. All data are presented as means ± SE (n = 5 in each group). Citrate Synthase activity was measured according to (De Lisio et al. 2011, Muscle Nerve 43: 58--64), and mitochondrial density was assessed according to (Idell-Wenger et al. 1978, J. Biol. Chem. 253: 4310--4318). Table S3. Oxidative stress parameters (carbonyl groups and lipid dienes (Oxidation Index (233/215)) in cardiac homogenates of C, E, P and S groups of rats. The mean values ± SE are expressed as percentage of the relevant control values. *P < 0.01, significantly different from the C, P and S groups (n = 6 in each group). Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article." @default.
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• W1592611595 title "Exercise-induced heart mitochondrial cholesterol depletion influences the inhibition of mitochondrial swelling" @default.
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• W1592611595 doi "https://doi.org/10.1113/expphysiol.2013.073007" @default.
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