Myoblasts were being processed forEL-102 (immune) electron microscopy as explained in resources and techniques. Representative electron micrographs demonstrate the morphology of caveolae (top panel) and caveolin 1 enriched “open” vesicular constructions at the plasma membrane (middle) or “closed” in the cytosol (base panel) (C). Scale bar = 100 nm particularly handle the influence of H2O2 on membrane caveolae, we quantified the relative abundance of caveolin one by TIRF microscopy. As proven in Fig. 4B and C, oxidative strain experienced no substantial influence on caveolin 1 distribution at the plasma membrane of myoblasts. According to these data, even though H2O2 remedy diminished caveolin 1 level, it had no considerable result on caveolae assembly and membrane relative distribution in C2C12 myoblasts.Effect of H2O2 on caveolin 1 localization. Cells ended up treated with 1 mM H2O2 throughout 1h. Caveolin 1 localization in C2C12 cells was decided by immunofluorescence labelling (A) or TIRF imaging (B). The ratio involving membrane place with caveolin one labeling and full membrane spot was expressed right after TIRF photographs evaluation (C). Bars on the graph symbolize the SEM. Scale bar = ten m.To analyze the outcome of H2O2 cure on caveolae-distinct endocytosis in C2C12 myoblasts, the cellular uptake of fluorescent Bodipy Lactosylceramide was quantified by circulation cytometry result of H2O2 on caveolae-dependent endocytosis. Cells were incubated with .twenty five M of Bodipylactosylceramide soon after treatments with H2O2 or distinct inhibitors of endocytic pathways as explained in materials and techniques. Cells ended up analyzed by move cytometry (exc = 488 nm) like at minimum ten 000 cells by situation (A). The imply mobile fluorescence depth was expressed as a share of the fluorescence calculated in the untreated management cells (—) (B and C). Bars on the graph signify the SEM. Drastically unique from the regulate sample (P < 0.01)analysis. Lactosylceramides have been shown to be specifically internalized in cells by caveolae [36, 37]. As shown in Fig. 5C, we confirmed that genistein, which inhibits caveolae-dependent endocytosis [38], significantly reduced Bodipy LacCer internalization. Chlorpromazine, an inhibitor of clathrin-dependent endocytosis, had no effect on intracellular fluorescence intensity. Cytochalasin D however, known to impair actin dynamics and thereby caveolae endocytosis, partially inhibited lactosylceramide uptake (Fig. 5C, 63% of the control). As expected, when all energy-dependent mechanisms were switched-down by ATP depletion (see "Deoxy" condition Fig. 5C), lactosylceramide uptake was very low as compared to untreated cells. After H2O2 addition, the intracellular relative fluorescence intensity was significantly lowered (75 and 43% of the controls after addition of 500 and 1000 M H2O2, respectively, Fig. 5). These data indicate a net decrease of caveolae-dependent endocytosis of the fluorescent sphingolipid. According to Sinha and co-workers, caveolae would be responsible for membrane tension buffering under hypo-osmotic conditions [15]. We thus submitted myoblasts to hypo-osmotic shock (30 mOsm) and measured membrane rupture 30 min later by Trypan blue staining. As soon as 5 min after changing the osmolarity in the culture medium, cell swelling could be easily observed by light microscopy (compare Iso and Hypo, Fig. 6A). The resulting increase in membrane tension led to the rupture of myoblast membranes in 30 minutes (Fig. 6B). Under milder condition (150 mOsm), cell swelling could be followed under the light microscope without effect of H2O2 on cell membrane rupture in hypo-osmotic conditions. Myoblasts were grown in iso- (300 mOsm) or hypo-osmotic medium (30 mOsm). Cell swelling was observed by light microscopy after 5 min (A). Cells were pre-incubated (or not) with H2O2 or -methyl-cyclodextrin (-MCD), left in iso- or hypoosmotic medium for 30 min and stained with Trypan Blue (B). The cells were treated with a caveolin 1 siRNA or a negative control as described in material and methods. The reduction of caveolin 1 expression was confirmed by western blot (C) and the above swelling protocol was applied to the cells pre-incubated (or not) with 500 M H2O2 (D). A minimum of 100 cells were counted by experiment and the Trypan blue positive cells were expressed as a percentage of the cells submitted to hypo-osmotic stress (-- in B and D). Bars on the graph represent the SEM. Significantly different from the relative iso-osmotic sample (P < 0.05). Significantly different from the untreated related control (without H2O2, P < 0.05). Significantly different from the negative control (P < 0.05). Scale bar = 30 m causing any significant damage on myoblast membranes (data not shown). When oxidative stress was applied to the cells 10 min beforehand, membrane rupture was significantly increased after 30 min in 30 mOsm medium (Fig. 6B). As a positive control cells were treated with -methylcyclodextrin (-MCD), a drug widely used to deplete cholesterol and disrupt caveolae structures [39]. As shown in Fig. 6, -MCD treatment significantly impaired cell resistance to osmotic shock in a similar way than H2O2 treatment. Although -MCD can impair caveolae formation, the drug might have a more pleiotropic effect in the cells. Therefore we used a specific siRNA and significantly reduced caveolin 1 expression (80% inhibition, Fig. 6C). As shown in Fig. 6D, in these conditions, the cells were less resistant to membrane tension increase. Both of the abovementioned experiments indicated that oxidative stress induction impairs two caveolae-dependent cell processes: endocytosis and membrane resistance to cell swelling.In this paper, we show for the first time that caveolin 1 is degraded by the proteasome after oxidative stress induction in mouse myoblasts. Oxidative damage has been proposed as one of the major contributors to skeletal muscle aging. When localized in satellite cells, it could be responsible for the failure of myogenic regenerative process observed during muscle ageing [40]. Caveolin 1 has been linked to oxidative-regulating pathways. The protein interaction with several oxidative enzymes (eNOS, NOX) is known to inhibit their specific activities [41]. More recently, several authors suggested that caveolin 1 could be responsible for negatively regulating antioxidant defenses in fibroblasts through its interaction with Nrf2 transcription factor [20, 42]. However, the effect of oxidative stress on the protein expression level itself still remained controversial and clearly dependent on the cell type. While Hsieh et al. showed that H2O2 treatment decreased caveolin 1 expression in cardiomyocytes, Dasari et al. demonstrated the opposite effect of comparable doses of oxidant in epithelial cells [43, 44]. Here, we added 500 M or 1000 M H2O2 directly to the culture medium of myoblasts as currently done [29, 45]. H2O2 is known to freely diffuse across cellular membranes and give rise to intracellular reactive oxygen species especially in skeletal muscle [46]. Nonetheless we controlled the induction of oxidative stress inside our cells by measuring ROS accumulation and their consequences on proteins. ROS significantly accumulated inside the cells as soon as 10 min after H2O2 addition to the culture medium. Moreover, protein carbonyls, one of the most current cellular consequences of oxidative stress [30], increased 3 to 4 times after H2O2 addition (Table 2). In a previous study carried out in rat skeletal muscle, we described similar increases in carbonyl content during aging [47]. Our conditions of treatment thus represent a good cellular model for studying the mechanisms involved in skeletal muscle aging. Interestingly, we were able to induce a significant oxidative stress in proliferating myoblasts without affecting the viability of the cells (see Table 1, 500 M 10 min to 3h, and 1000 M 10 min). Most importantly, caveolin 1 expression level was very rapidly affected after H2O2 addition to the culture medium (10 min, Fig. 1). The protein level decreased by almost 30%, similarly to what Hsieh et al. recently described in cardiomyocytes [43]. These results suggest that the protein is directly, but most remarkably, very quickly targeted by oxidative stress in proliferating mouse myoblasts. According to several authors, such a rapid elimination of a protein could be imputable to the proteasome, especially after oxidative injuries [31, 48]. Here we showed that H2O2 had no longer effect on caveolin 1 expression level when cells were pre-incubated with a specific proteasome inhibitor (MG132) (Fig. 2). These data confirm that caveolin 1 is rapidly degraded by the proteasome-dependent pathway after H2O2 treatment of the cells. As aforementioned, caveolin 1 would behave as a negative regulator of cellular antioxidant defenses. In the light of the present data, the early degradation of the protein after stress induction could then trigger the cellular antioxidant defenses. During skeletal muscle differentiation and/or regeneration, caveolin 1 and 3 are sequentially expressed [49]. Caveolin 1 is highly expressed in proliferating myoblasts and cav-1 gene becomes switched-off as the cells begin to fuse into multinucleated myotubes. In parallel, caveolin 3 expression is up-regulated as differentiation of the myogenic cells occurs. Both proteins are however absolutely necessary for caveolae biogenesis and structuration [50, 51]. Mutations in the cav-3 gene or alterations of caveolin 3 expression level have been closely associated with major skeletal muscle dysfunctions and pathologies (LGMD1C, DMD, . . .) [17, 18]. On the opposite, the consequences of caveolin 1 deregulation has never been clearly studied in muscle, although the protein has been largely linked to several major diseases in other tissues: breast cancer, atherosclerosis. . .[52, 53]. Here we observed a significant loss of caveolin 1 in proliferating myoblasts after intracellular stress induction. We next asked whether the decrease in caveolin 1 would lead to a loss of caveolae. Surprisingly, we were not able to evidence any effect of H2O2 treatment on caveolae membrane density by TEM (Fig. 3), as shown before in cardiac myocytes for instance [54]. Caveolae were however too sparse at the membrane in our cell type compared to cardiac tissue. We therefore combined biochemical and microscopic approaches to access the information. First, we looked at the ability of caveolae to assemble normally in cells submitted to oxidative stress. Caveolin-containg fractions were isolated by sucrose density fractionation from myoblasts treated with H2O2. As shown in Fig. 3A, caveolin 1 was enriched in low-density fractions (2 and 3) of the gradient, as confirmed by the co-localization of caveolin 1 and cavin 1. Remarkably, H2O2 addition to the cells did not significantly affect the fractionation of caveolin 1 between low- and high-density fractions. Whereas these data cannot rule out that the caveolae relative amount was unaffected by H2O2, they suggest however that caveolae were still able to assemble normally regardless of H2O2 addition. Cellular material obtained in fraction 2 was also observed by TEM after caveolin 1 specific labelling. As clearly seen on the pictures, H2O2 did not affect the size of the vesicles, neither changed the relative abundance of caveolin 1 per caveolae (Fig. 3B). Second, TIRF microscopy was carried out to follow the effect of oxidative stress on caveolin 1 relative abundance at the plasma membrane. The specific fluorescence of the protein detected at the membrane of the myoblasts stayed unchanged whether the cells were treated with 1 mM H2O2 or not (Fig. 4). The discrepancy between the strong signal monitored by TIRF microscopy and the few caveolae observed in C2C12 myoblasts by TEM could be explained by the conformation of the caveolae itself. Although caveolae can be present at the plasma membrane, they might require an activation step to harbor the omega shape classically detected by TEM [55]. Altogether these data indicate that H2O2-induced caveolin 1 degradation had no significant consequence on caveolae assembly and localization at the plasma membrane. We next focused on the consequences of H2O2 treatment on caveolae-mediated functions. We studied two caveolae-dependent cell functions: endocytosis and mechanosensing [12, 35]. Lactosyl-ceramides have been shown to be internalized by caveolae [36, 37]. We therefore monitored caveolae endocytosis with this specific probe in an assay where internalization of the fluorescent Bodipy coupled lactosyl-ceramide was measured by flow cytometry analysis after H2O2 treatment. The data nicely showed a net decrease of the intracellular fluorescence as H2O2 increased (Fig. 5).24805071 As shown in Fig. 5C, when caveolae-specific endocytosis was inhibited (genistein), the fluorescence intensity significantly decreased in the cells, confirming the specificity of the assay. We also blocked actin dynamics with cytochalasin D, and we observed a partial inhibition of the ceramide uptake (Fig. 5C) in agreement with published data on cytochalasin D impairing caveolae internalization through actin depolymerization [56]. According to our data, oxidative stress induction is likely to negatively impact caveolar endocytosis. Another function of caveolae has recently emerged in the literature: the maintenance of plasma membrane through the constitution of a caveolae “membrane reservoir” buffering membrane tension variations [15, 54]. In our experiments, we studied the effect of oxidative stress on membrane tension buffering under hypo-osmotic conditions. Proliferating myoblasts were submitted to hypo-osmotic shock and membrane rupture was measured by the penetration of Trypan blue dye. As shown in Fig. 6, reducing the osmolarity in the culture medium from 300 to 30 mOsm caused the rupture of myoblast membranes in 30 min. In another experiment, we incubated the cells in a 150 mOsm medium and had no effect on plasma membranes, although the cell swelling could be microscopically observed (data not shown). As mentioned above, caveolae are able to supply the cells with “new” membranes when necessary, we could therefore imagine that 30 mOsm would overcome the ability of caveolae to maintain cell membrane integrity. When H2O2 was applied to the cells 10 min beforehand, membrane rupture was significantly increased after 30 min in 30 mOsm medium suggesting that caveolae integrity and/or number were affected and could not guarantee mechanical resistance anymore (Fig. 6B). Similar result was obtained with methyl-beta-cyclodextrin, a cholesterol-complexing drug currently employed to disrupt caveolae inside the cells [39, 54], corroborating the role of caveolae in buffering membrane tension. To further confirm the effect observed with methylbeta-cyclodextrin, we directly inhibited caveolin 1 expression with siRNA to have a more specific control. As shown in Fig. 6C, we reduced caveolin 1 expression by 80%. In this condition, myoblasts were three times more sensitive to the hypo-osmotic shock (Fig. 6D). Remarkably, when H2O2 was added during the osmotic stress, the amount of Trypan blue positive cells stayed unchanged indicating that osmotic swelling-induced membrane rupture was only imputable to a breach in caveolae.