Simvastatin inhibits phosphorylation of Akt Ser473 and S6rp in C2C12 cells and mouse skeletal muscle.
As shown in Fig. 1, treatment with simvastatin inhibited the phosphorylation of Akt Ser473 and S6rp in C2C12 myoblasts (Fig. 1A), C2C12 myotubes (Fig. 1B) and in mouse skeletal muscle (Fig. 1C). These findings show that simvastatin inhibits the activity of mTORC1 (inhibition of S6rp phosphorylation) and of mTORC2 (inhibition of Akt Ser473 phosphorylation) in vitro and in vivo. In the following, C2C12 myoblasts and myotubes were used to find out the mechanisms of simvastatin-associated mTORC inhibition and myotoxicity.
Rapamycin inhibits mTORC1 in C2C12 myotubes but is not cytotoxic and does not affect Akt Ser473 phosphorylation.
We first compared simvastatin-induced toxicity and the toxic effects of specific mTORC1 inhibition on C2C12 cells. We treated C2C12 myotubes with 100 nM rapamycin, a specific inhibitor of mTORC1, to investigate membrane toxicity and ATP content (Fig. 2A and 2B). Simvastatin led to a significant 2-fold increase in AK release (reflecting disturbed membrane integrity) and to a concordant depletion of intracellular ATP (Fig. 2A and 2B). Insulin prevented the toxic effects of simvastatin in co-treatment (Fig. 2A and 2B). Interestingly, and in contrast to simvastatin, rapamycin decreased AK release alone or in-co-treatment with simvastatin and/or insulin (Fig. 2A and 2B). To confirm that mTORC1 was inhibited, we quantified the phosphorylation of the S6rp, a marker of mTORC1 activity. The phosphorylation of S6rp (Ser235/236) was almost completely abolished in all incubations containing rapamycin (Fig. 2C). As established previously [14], also simvastatin significantly decreased the phosphorylation of the S6rp (Fig. 2C), but to a lesser extent than rapamycin (Fig. 2C). To determine if inhibition of mTORC1 influences the activity of mTORC2, we analyzed the phosphorylation of Akt Ser473, a substrate of mTORC2. Whereas simvastatin decreased Akt Ser473 phosphorylation, rapamycin alone did not affect the phosphorylation of Akt Ser473 (Fig. 2D). Co-treatment with simvastatin and rapamycin decreased the phosphorylation of Akt Ser473, whereas in the presence of the combination insulin and rapamycin, Akt Ser473 phosphorylation was maintained (Fig. 2D).
mTOR is a protein kinase that can be phosphorylated and is a component of both mTORC1 and mTORC2. As shown in supplementary Fig. 1 (Fig. S1), rapamycin impaired Ser2448 phosphorylation of mTOR to a larger extent than simvastatin, whereas insulin did not affect mTOR phosphorylation. Interestingly, simvastatin mitigated the effect of rapamycin on Ser2448 mTOR phosphorylation, suggesting an interaction between the two compounds.
Rheb is a small GTPase, which is membrane-anchored and therefore prenylated, and which is important for the activation of mTORC1 [27–30]. As shown in supplementary Fig. 2A (Fig. S2A), simvastatin, but not rapamycin or insulin, impaired the prenylation of Rheb. In contrast, mRNA expression of Rheb was not significantly affected by simvastatin or rapamycin (Fig. S2A).
These data demonstrate that, in contrast to simvastatin, specific mTORC1 inhibition with rapamycin did neither induce myotoxicity, nor reduce mTORC2 activity or impair Rheb prenylation. Simvastatin inhibited mTORC1 by impairing the phosphorylation of Akt Ser473 and Rheb prenylation. However, the cytotoxicity of simvastatin could not be explained by inhibition of mTORC1.
mTORC2 inactivation in C2C12 myoblasts leads to a similar toxicity and impairment of Akt signaling as treatment with simvastatin.
We showed in several studies that simvastatin significantly and strongly inhibits the phosphorylation of Akt at the serine 473 site [12–15]. Since Akt Ser473 phosphorylation is performed by mTORC2, we hypothesized that mTORC2 inhibition might be the primary event in simvastatin-induced muscle damage and myocyte cell death. To test this hypothesis, we investigated whether mTORC2 inactivation could induce similar toxic effects than those observed with simvastatin. Using RNA interference, we knocked down Rictor to abrogate mTORC2 activity. siRNA targeted against Rictor decreased Rictor gene and protein expression in C2C12 myoblasts, while control siRNA did not affect Rictor expression (Fig. 3C; supplementary Fig. 3A and 3B). The transfection was efficient and stable for at least 72 hours on the protein level.
We then evaluated the effects of mTORC2 inhibition on membrane integrity (AK release into the medium) and on the cellular ATP content. Cells with Rictor knock-down treated with DMSO displayed similar toxicities (AK release and ATP content) as simvastatin-treated control cells (Fig. 3A and 3B). Control siRNA-transfected cells showed a significantly higher AK release with simvastatin (10-fold increase), simvastatin and insulin (7-fold-increase), and, in a less pronounced manner, simvastatin and 100 µM mevalonate (5-fold increase) compared to DMSO-treated cells (Fig. 3A). Similar effects were observed on the ATP content of the C2C12 myoblasts (Fig. 3B). Insulin, mevalonate, geranylgeraniol and farnesol applied alone did not induce toxic effects in control-transfected cells (Fig. 3A and 3B). Geranylgeraniol prevented the effects of simvastatin completely (SMV + GGOH 50 µM), whereas farnesol and mevalonate were less effective in preventing the toxicity of simvastatin (Fig. 3A and 3B). In contrast, the addition of mevalonate, geranylgeraniol or farnesol did not prevent the toxicity associated with Rictor knock-down (Fig. 3A and 3B). Simvastatin increased the toxicity of Rictor knockdown, but in a less than additive manner (Fig. 3A and 3B).
To find out whether mTORC2 knock-down leads to similar defects on Akt signal transduction than simvastatin, we investigated the activation of Akt (phosphorylation on serine 473) and of the downstream effector S6rp. As shown in Fig. 3C-E, phosphorylation of Akt Ser473 was significantly decreased in Rictor siRNA-transfected compared to control cells, showing that mTORC2 is responsible for Akt Ser473 phosphorylation. Phosphorylation levels of Akt Ser473 were similar between simvastatin-treated control cells and C2C12 cells with Rictor knock-down, and simvastatin increased the inhibition of the phosphorylation of Akt Ser 473 when Rictor was absent. Mevalonate and geranylgeraniol maintained Akt Ser 473 phosphorylation in presence of simvastatin, while they were ineffective in cells with Rictor knock-down. Interestingly, insulin was able to maintain Akt Ser473 phosphorylation in Rictor knock-down cells, possibly due to remaining mTORC2 activity. Similar effects were observed for the phosphorylated form of the S6rp, a downstream effector of Akt and mTORC1.
Since mTORC2 stimulates cellular proliferation [36], we also assessed the effect of Rictor siRNA transfection and simvastatin on Ki-67 mRNA expression, which is a marker of cell proliferation [42]. As shown in supplementary Figure S4, both simvastatin and Rictor knockdown caused a dramatic decrease in Ki-67 mRNA expression, indicating impaired C2C12 myoblast proliferation. Insulin could not prevent this decrease, whereas mevalonate partially prevented the decrease in Ki-67 mRNA associated with simvastatin, but not with Rictor knockdown.
These results clearly show that most toxic effects and most effects on Akt signal transduction of simvastatin in C2C12 cells can be explained by inhibition of mTORC2. The data also indicate that the mechanisms of mTORC2 inhibition involves prenylation defects since replenishment with isoprenoids could prevent the cytotoxicity associated with simvastatin, but not the cytotoxicity associated with Rictor knock-down.
mTORC2 inactivation in simvastatin-treated myotubes is independent of Ras prenylation.
Since the addition of isoprenoids prevented cytotoxicity and impaired Akt activation by simvastatin, we investigated the role of proteins needing prenylation as a reason for impaired mTORC2 activity caused by simvastatin. Prenylation is performed with two products of the mevalonate pathway, geranylgeranyl pyrophosphate and farnesyl pyrophosphate, and up to 2% of cellular proteins are modified with this lipid post-translational modification [43]. Prenylation is necessary for certain proteins that need attachment to cellular membranes to be active. The investigation of simvastatin’s effects on cell viability and Akt phosphorylation shown Fig. 3 revealed that mainly geranylgeraniol could prevent cytotoxicity and impaired activation of mTORC2, indicating that diminished prenylation is a probable mechanism contributing to toxic effects of simvastatin in skeletal muscle cells.
To assess this possibility, we first examined the prenylation of Ras. Ras is a prenylated small GTPase that activates the PI3K pathway and promotes proper mTORC2 activation [33; 44]. Cells treated with simvastatin displayed impaired Ras prenylation (Fig. 4A), as demonstrated by an increased unprenylated fraction of this GTPase. Neither mevalonate and nor geranylgeraniol restored Ras prenylation in the presence of simvastatin (Fig. 4A). However, geranylgeraniol completely prevented the decrease in Akt Ser473 phosphorylation (reflecting mTORC2 activity) in C2C12 myotubes treated with simvastatin (Fig. 4B), suggesting that inhibition of Ras prenylation by simvastatin is independent of impaired Akt Ser473 phosphorylation. As the addition of mevalonate and geranylgeraniol did not restore Ras prenylation in the presence of simvastatin, we assessed the effect of farnesol on Ras. After 24 hours of incubation, farnesol did not prevent impaired prenylation of Ras by simvastatin (Fig. 4C), nor did it improve impaired P-Akt Ser473 phosphorylation (Fig. 4D). However, after 48 hours of incubation, farnesol increased prenylation of Ras (Fig. 4C), but still failed to improve the phosphorylation of Akt Ser473 (Fig. 4D).
These results indicate that geranylgeraniol, but not farnesol, can prevent the effect of simvastatin on Akt phosphorylation but in a manner that is independent of Ras prenylation.
Geranylgeraniol enables prenylation of Rap1 in the presence of simvastatin and prevents cytotoxicity associated with simvastatin.
Rap1 is a small GTPase that has been shown to regulate the activity of mTORC2 in the amoeba Dictyostelium discoideum (Khanna, 2014). Considering its role in mTORC2 activation in amoeba, we investigated a possible function of Rap1 in the activation of mTORC2 and in the toxicity of simvastatin on C2C12 myoblasts and myotubes. As shown in Fig. 5A and 5B, simvastatin inhibited the prenylation of Rap1 efficiently. This effect could not be prevented by the addition of mevalonate, but by 50 µM geranylgeraniol. A lower geranylgeraniol concentration (10 µM) decreased the non-prenylated Rap1 fraction numerically, without reaching statistical significance. Mevalonate was not able to prevent the cytotoxicity of simvastatin completely, whereas geranylgeraniol was protective already at 10 µM (Fig. 5C).
To study the role of Rap1 in simvastatin-associated cytotoxicity in more detail, we performed a knock-down of Rap1 in C2C12 myoblasts using a siRNA approach (Fig. 5D). Rap1 knock-down was associated with a numerical increase in AK release and ATP depletion, which was augmented by simvastatin (Fig. 5E and 5F). The addition of mevalonate prevented the effect of simvastatin on AK release completely and of the combination simvastatin/Rap1 knock-down partially. In comparison, the addition of geranylgeraniol prevented the effect of simvastatin and the combination simvastatin/Rap1 knock-down on AK release and ATP depletion completely.
These results support a role of Rap1 in the toxicity of simvastatin on C2C12 cells and indicate that prenylation plays a role in the function of Rap1.
Rap1 knock-down causes similar effects on the Akt pathway, atrogin-1 mRNA and Ki-67 mRNA expression as simvastatin.
Next, we assessed the role of Rap1 in the function of mTORC1 and mTORC2 and in the downstream effects. Knock-down of Rap1 was associated with a clear drop in the Akt Ser473 phosphorylation, proving the importance of Rap1 for mTORC2 activation (Fig. 6A). Simvastatin showed the expected decrease in Akt Ser473 phosphorylation and increased the effect of Rap1 knock-down. The addition of mevalonate partially and of geranylgeraniol completely prevented the effect of simvastatin on Akt Ser473 phosphorylation.
In addition, treatment with simvastatin and Rap1 knock-down were associated with an increase in atrogin-1 mRNA expression and a decrease in the mRNA expression of Ki-67 (supplementary Fig. S5). Atrogin-1 belongs to the F-box protein family and forms one of four subunits of the ubiquitin protein ligase complex SCFs [45]. Atrogin-1 expression can be regarded as a marker of muscle atrophy and has been shown to be increased in C2C12 cells and mice exposed to simvastatin [46–48]. Mevalonate and geranylgeraniol prevented the increase in atrogin-1 mRNA in simvastatin-treated C2C12 myoblasts, but not in cells with Rap1 knock-down (supplementary Fig. S5A). In addition, mevalonate prevented the mRNA decrease in Ki-67 partially and geranylgeraniol completely in C2C12 myoblasts treated with simvastatin, but not in cells with Rap1 knock-down (supplementary Fig. S5B).
The experiments show that Rap1 activates mTORC2 and support the role of Rap1 in the toxicity of simvastatin on C2C12 cells.
Simvastatin increases mitochondrial ROS production which can be mitigated by antioxidants and which contributes to impaired function of mTORC2 and cytotoxicity.
The lower cytotoxicity of Rap1 knockdown compared to simvastatin (Fig. 5D and 5E) despite similar effects on the mTORC2/Akt pathway (Fig. 6B) suggested that the cytotoxicity of simvastatin was not only due to inhibition of mTORC2 but could have additional components. We and others have shown repeatedly that statins impair the function of the mitochondrial electron transport chain [46; 49; 50], which is associated with mitochondrial ROS production [40]. As shown in Fig. 7A, mitochondrial accumulation of ROS was increased in C2C12 myoblasts treated with simvastatin, whereas Rap1 knock-down was not associated with mitochondrial ROS accumulation. Geranylgeraniol prevented ROS accumulation completely, indicating that geranylgeraniol can act as a ROS scavenger. As expected, also the complex III inhibitor antimycin A was associated with mitochondrial ROS accumulation, which could be prevented by geranylgeraniol or by the antioxidant MitoTEMPO. Since mitochondrial ROS accumulation is often associated with increased SOD2 expression [51; 52], we determined SOD2 protein expression by western blotting. As shown in Fig. 7B, simvastatin and Rap1 knockdown increased SOD2 protein expression numerically compared to control siRNA treated cells, whereas the combination simvastatin and Rap1 knockdown increased SOD2 expression significantly. This was partially prevented by mevalonate and completely by geranylgeraniol. Next, we aimed to find out whether mitochondrial dysfunction contributes to simvastatin-associated cytotoxicity and inhibition of mTORC2 in C2C12 myotubes. As shown in Fig. 7C, simvastatin and antimycin A increased mitochondrial ROS accumulation also in C2C12 myotubes, which could be prevented by MitoTEMPO. As expected, simvastatin and antimycin A caused AK release from C2C12 myotubes, which could be prevented by the addition of MitoTEMPO (Fig. 7D). Similarly, simvastatin and antimycin A caused a drop in the ATP content of C2C12 myotubes (Fig. 7E). This drop could be prevented by MitoTEMPO for simvastatin, but not for antimycin A. Simvastatin and antimycin A impaired Akt Ser473 phosphorylation, which could be partially (simvastatin) or completely (antimycin A) be prevented by MitoTEMPO (Fig. 7F).
These results show that mTORC2 can also be inhibited by mitochondrial ROS accumulation and that mitochondrial dysfunction contributes to the cytotoxicity of simvastatin.