The reduction in cholesterol in mitochondria, observed after exercise, is related to the inhibition of mitochondrial swelling. Caveolin-1 (Cav-1) plays an essential role in the regulation of cellular cholesterol metabolism and is required by various signalling pathways. Therefore, the aim of this study was to investigate the effect of prolonged swimming on the mitochondrial Cav-1 concentration; additionally, we identified the results of these changes as they relate to the induction of changes in the mitochondrial swelling and cholesterol in rat skeletal muscle and liver. Male Wistar rats were divided into a sedentary control group and an exercise group. The exercised rats swam for 3 hours and were burdened with an additional 3% of their body weight. After the cessation of exercise, their quadriceps femoris muscles and livers were immediately removed for experimentation. The exercise protocol caused an increase in the Cav-1 concentration in crude muscle mitochondria; this was related to a reduction in the cholesterol level and an inhibition of mitochondrial swelling. There were no changes in rat livers, with the exception of increased markers of oxidative stress in mitochondria. These data indicate the possible role of Cav-1 in the adaptive change in the rat muscle mitochondria following exercise.
The significance of the exercise-induced depletion in the mitochondrial cholesterol pool is still not fully understood. The reduction in cholesterol in rat heart mitochondria, observed after exercise, is related to the inhibition of mitochondrial swelling; however, this change does not influence the mitochondrial bioenergetics and oxidative stress. [
Mitochondrial swelling [
Caveolins (Cav) are essential components of caveolae, which are plasma membrane invaginations that demonstrate reduced fluidity; this reflects an accumulation of cholesterol [
Cav-1 deficiency is related to impaired mitochondrial function, free cholesterol accumulation in mitochondrial membranes, increased membrane condensation, a reduction of the respiratory chain, and accumulation of reactive oxygen species [
However, to our knowledge, there are no data explaining the mechanism responsible for the cholesterol depletion in mitochondria following exercise. Therefore, the aim of this study was to investigate the effect of prolonged swimming on mitochondrial Cav-1 and cholesterol concentrations as well as the induction of changes in mitochondrial swelling in rat skeletal muscles and livers. Based on our previous data [
All chemicals were purchased from Sigma (St. Louis, MO, USA), with the exception of BSA (Merck, Darmstadt, Germany). The cholesterol assay kit was generously donated by CHEMA Diagnostica (Monsano, Italy).
Male Wistar rats (
The rats were prepared for the experiments and exercise tests using the previously described methods [
The liver mitochondria were isolated, as previously described by Broekemeier et al. [
The skeletal muscle mitochondria were isolated, as previously described by Fontaine et al. [
All steps were performed at 4°C.
The measurement of RLM and RQM swelling was spectrophotometrically performed, as previously described for the liver by Crouser et al. [
CaCl2 (100
The swelling curves were recorded at 540 nm. Cuvettes, containing the mitochondrial suspension, were kept at 25°C.
The greater decrease in absorbance is related to greater susceptibility of mitochondria to calcium chloride-induced mitochondrial swelling. Such deenergized and inactivated mitochondria are rapidly and physiologically less resistant to opening mPTP, which precedes the process of cell death.
Total lipids were extracted from the mitochondria normalized to the mitochondrial protein concentration. The cholesterol content in extracts was measured using the CHEMA Diagnostica cholesterol assay kit with a standard cholesterol solution as a reference. Total lipids extraction and cholesterol measurement procedures were performed as previously described [
The concentration of caveolin-1 was measured using the Cloud-Clone Corporation Caveolin-1 ELISA Kit (Category number SEA214Ra) according to the manufacturer’s instructions.
The carbonyl groups [
The protein content was measured using the method described by Lowry et al. [
Statistical analyses were performed using a software package (Statistica v. 10.0, StatSoft Inc., Tulsa, OK, USA). The results are expressed as the mean ± standard error (SE). The differences between the means were tested using the unpaired Student’s
The prolonged swimming protocol caused a significant drop in the cholesterol level in crude mitochondria of the quadriceps muscle. The mitochondria isolated from the skeletal muscle of exercised rats had approximately 84% of the cholesterol compared with control rats (Figure
Mitochondrial cholesterol levels in controlled and exercised animals. Cholesterol was measured in crude mitochondria isolated from the quadriceps (a) and liver (b). The data are presented as the means ± SE.
We determined whether mitochondrial cholesterol depletion was associated with mitochondrial swelling. The calcium chloride-induced mitochondrial swelling was significantly lower in the rat quadriceps muscle of mitochondria isolated after exercise when compared with the control (Figure
Mitochondrial swelling in controlled and exercised animals. The calcium chloride-induced mitochondrial swelling was spectrophotometrically assessed in the quadriceps (a) and liver (b) crude mitochondria. The data are presented as the means ± SE.
To verify whether the depletion of the mitochondrial cholesterol level after exercise is related to changes in Cav, the concentration of Cav-1 was determined in crude mitochondria of the C and E groups.
Prolonged swimming caused a significant increase in the content of Cav-1 in the quadriceps of crude mitochondria (Figure
Mitochondrial caveolin-1 levels in controlled and exercised animals. Caveolin-1 level was measured in crude mitochondria isolated from the quadriceps (a) and liver (b). The data are presented as the means ± SE.
Correlation between caveolin-1 and cholesterol levels in quadriceps crude mitochondria. A Pearson product-moment correlation coefficient was computed to assess the relationship between the caveolin-1 and cholesterol level in the crude mitochondria isolated from the quadriceps. There was a large, negative correlation between the two variables,
To verify that exercise was able to induce oxidative stress, protein carbonyl groups and lipid dienes were measured in the liver and quadriceps muscle crude mitochondria of the C and E groups of rats. Significantly higher oxidative stress parameters were only observed in the E group of crude mitochondria in the liver (Table
Oxidative stress parameters (carbonyl groups and lipid dienes) in Quadriceps and Liver mitochondria of the C and E groups of rats.
Quadriceps mitochondria | Liver mitochondria | |||
---|---|---|---|---|
C | E | C | E | |
Carbonyl groups (nmol (mg of protein)−1) | 1.460 ± 0.08 | 1.364 ± 0.12 | 1.125 ± 0.07 | 1.732 ± 0.12 |
Lipid dienes (oxidation index (233/215)) | 0.172 ± 0.003 | 0.169 ± 0.004 | 0.230 ± 0.005 | 0.260 ± 0.005# |
The mean values ± SE are expressed as nmol (mg of protein)−1 (for carbonyl groups) and oxidation index 233/215 (lipid dienes).
In the present study, we demonstrate that adaptive responses to prolonged exercise are related to increased content of Cav-1 in isolated quadriceps muscle mitochondria; moreover, there is concomitant depletion of mitochondrial cholesterol and increased resistance to Ca2+ induced swelling. We did not observe similar changes in rat liver mitochondria from the exercised group, but we did observe an increase in the level of protein carbonyl groups and lipid dienes in mitochondria (markers of oxidative stress). There was an inverse correlation between mitochondrial cholesterol and Cav-1 levels in rat tissues after exercise. The data confirm our initial hypothesis that both exercise-induced changes in mitochondrial swelling and the level of cholesterol are related to increases in Cav-1 concentrations in skeletal muscle mitochondria but not in the liver. We suspect that this phenomenon refers exclusively to the contractile tissues, such as heart and skeletal muscles. The finding implies that a reduction in the cholesterol levels and an increased mitochondria sensitivity to swelling are not deleterious; furthermore, these alterations may be a result of changes in Cav-1 concentrations in the mitochondria. As we have previously indicated, these modifications are related to a dynamic physiological process that may help mitochondria adapt to stress conditions [
To our knowledge, there are no data that show changes in cholesterol and Cav-1 levels in liver and skeletal muscle mitochondria after exercise. However, Park and colleagues showed that there are changes in the Cav-1 concentrations in skeletal muscles [
Our data demonstrate that prolonged swimming reduced cholesterol levels and increased Cav-1 concentrations in rat skeletal muscle mitochondria. We did not observe such changes in the hepatic mitochondria, which leads us to suspect that this effect is specific to contractile tissues. Furthermore, there were exercise-induced changes in the mitochondrial Cav-1 and cholesterol content in skeletal muscles but not in the liver; this may have resulted from different responses in these tissues during oxidative stress.
Recently, we reported that modifications in mitochondrial function depend on the degree of depletion of cholesterol. This may be comparable to the changes observed after exercise. These changes did not impair the mitochondrial bioenergetics; furthermore, the changes positively inhibited the mitochondrial swelling, likely by remodelling the lipid microdomains [
It has been shown that mitochondria are cholesterol-sensitive organelles, but little is known about the regulation of their cholesterol influx/efflux [
There is an abundance of data which indicate that alternations in caveolin and cholesterol levels in the cell lead to oxidative stress. It was shown that elevated mitochondrial cholesterol levels lead to reduced 2-oxoglutarate transport and an impaired glutathione (GSH) import into mitochondria [
Studies in the changes in Cav-1 and cholesterol concentrations in mitochondria seem to be important for understanding the physiological and pathological mechanisms that occur in mitochondria in living organism.
The mechanism behind chronic exercise on the above phenomena remains to be resolved. Based on our previously published results and the results of others [
The Cav-1-dependent hypothesis of mitoprotection under exercise conditions. Under physiological conditions: appropriate levels of Cav-1 and cholesterol concentrations are presented in the mitochondria of contractile tissues. Under pathological conditions in crude mitochondria, decreased Cav-1, increased cholesterol concentrations, and mitochondrial swelling are observed. Exercise induces the opposite effects; for example, increased Cav-1 levels and decreased cholesterol levels in mitochondria lead to the inhibition of mitochondria swelling.
The presented data indicate exercise-induced modification in the mitochondrial cholesterol content specific to contractile tissue, which may have resulted from various response to oxidative stress. Moreover, the possible role of Cav-1 on mitochondrial cholesterol depletion following exercise may be fundamental for understanding the mitoprotective mechanism during stressful conditions. Further investigations are required to clarify the role of cholesterol and Cav-1 changes in mitochondria of different tissues following exercise and to explain whether exercise might reverse the mitochondria from a pathological to physiological state via a Cav-1-dependent mechanism.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Rosita Gabbianelli and Wieslaw Ziolkowski share senior coauthorship.
This study was supported by a Grant from the Polish Ministry of Science and Higher Education (no. N N404 168434) and from the National Science Centre in Poland (DEC-2013/09/N27/02538).