Simvastatin represses protein synthesis in the muscle-derived C₂C₁₂ cell line with a concomitant reduction in eukaryotic initiation factor 2B expression

Abstract

Statins are a widely prescribed class of cholesterol lowering drugs whose use is frequently associated with muscle-related ailments. A number of mechanisms have been implicated in statin-induced myotoxicity including alterations in both protein synthesis and protein degradation. The objective of the present study was to explore the mechanism(s) contributing to the statin-induced reduction in protein synthesis in the muscle-derived C₂C₁₂ cell line. Cells were treated with 10 μM simvastatin or vehicle alone for 24 h in 1% serum. Cells exposed to simvastatin exhibited reduced rates of protein synthesis, as evidenced by [³⁵S]methionine and [³⁵S]cysteine incorporation into protein. The reduction in protein synthesis occurred with a concomitant decrease in expression and activity of eukaryotic initiation factor 2B (eIF2B), a regulated and rate-controlling guanine nucleotide exchange factor known to affect global rates of protein synthesis. The reductions in protein synthesis and eIF2B expression were prevented by coincubation with mevalonate. Simvastatin treatment also resulted in a proteasome-sensitive reduction in the protein expression of all the subunits of the eIF2B heteropentameric complex. Finally, increased phosphorylation of the catalytic ϵ-subunit at Ser⁵³⁵ was observed, an event consistent with an observed reduction in eIF2B activity. These results suggest that repression of eIF2B expression and activity may contribute, at least in part, to the statin-induced reduction in protein synthesis.

statins [3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors] are a widely prescribed and effective class of lipid lowering drugs that have demonstrated enormous clinical benefit not only for reducing cardiovascular risk (26) but also for several additional and unexpected pleiotropic benefits (e.g., anti-inflammatory and immunomodulatory effects) (10, 23). One of the less desirable effects of statin use is the incidence of myopathies ranging from complaints of muscle pain and weakness (i.e., myalgia) to the most severe case of rhabdomyolysis, which is rare but can be fatal (2, 34). The mechanisms implicated in the myotoxic effects of statins are still poorly understood. Recently, statins have been shown to upregulate the “atrogenes” MAFbx and/or MuRF-1 in human, zebrafish, and rodent skeletal muscle, which may contribute to increased rates of muscle proteolysis (4, 11, 21). In addition to enhanced proteolysis, other groups have reported decrements in the rate of protein synthesis in embryonic chick cardiomyocytes (28) and rat primary myocytes (8, 22), although the mechanism for the repression of protein synthesis remains unclear.

One important point of regulation in the control of protein synthesis, particularly in the initiation phase of mRNA translation, occurs in response to altered activity and/or expression of eukaryotic initiation factor 2B (eIF2B). The eIF2B holoenzyme (consisting of five subunits: α, β, γ, δ, and ϵ) is regulated through a variety of mechanisms, and it functions as a guanine nucleotide exchange factor (GEF) for its substrate, eIF2. During the initiation phase of mRNA translation, proper assembly of the preinitiation complex requires eIF2 in a GTP-bound state to deliver the initiator methionyl tRNA (i.e., a ternary complex) to the 40S ribosomal subunit for processive scanning for the start codon of the mRNA. In response to a number of inhibitory (stress) signals, the activity of eIF2B, and thus the rate of assembly of ternary complex, is reduced, which can lead to the repression of global rates of protein synthesis. Intriguingly, there have been a number of reports indicating that statins attenuate or prevent cardiac hypertrophy (5, 20, 24, 43). In this context, it is important to note that eIF2Bϵ is implicated in β-adrenergic mediated cardiomyocyte hypertrophy (12) and that overexpression of eIF2Bϵ increases GEF activity and/or protein synthesis in a number of different cell types (3, 12, 35, 40, 42). In contrast, reductions in eIF2B expression and/or activity occur in rat skeletal muscle when protein synthesis and/or muscle growth is impaired (13, 38). Thus, the purpose of the current investigation was to further elucidate potential mechanisms of the statin-associated repression of protein synthesis. Specifically, we investigated the effects of simvastatin treatment on protein synthesis and eIF2B expression and in the muscle-derived C₂C₁₂ cell line.

MATERIALS AND METHODS

Cell culture.

Simvastatin (Sigma-Aldrich, St. Louis, MO) and MG-132 (CalBiochem/EMD Biosciences, La Jolla, CA) were prepared as 10 mM stock solutions in DMSO. Mevalonate was purchased as mevalonolactone (Wako Pure Chemical Industries, Osaka, Japan), prepared as a 0.5 M stock in water, and when utilized was added to the medium at a final concentration of 100 μM for the entire 24-h treatment period. C₂C₁₂ myoblasts (ATCC, Manassas, VA) were routinely maintained in growth medium (GM), consisting of Dulbecco's modified Eagle's medium (DMEM; GIBCO-Invitrogen, Carlsbad, CA) containing 25 mM glucose, 10% fetal bovine serum (FBS; Atlas Biologicals), and 1% penicillin-streptomycin (Invitrogen), at 37°C and 5% CO₂. Myoblasts were initially seeded at 3.75 × 10⁵ (control) or 5.0 × 10⁵ (simvastatin-treated) cells per 60-mm culture dish in GM for 16 h prior to treatment. In experiments utilizing myoblasts, cells were seeded at a higher density in the simvastatin-treated group on the basis of pilot experiments determining that cell confluence was more similar to the controls on the day of harvest. Myoblasts were differentiated into myotubes by seeding 5.0 × 10⁵ myoblasts per 60-mm dish and incubating in GM for 24 h followed by 3 days of incubation in serum-free DMEM with 1× ITS liquid media supplement (Sigma-Aldrich). On the day of the experiment, cells were washed once with serum-free DMEM and then maintained in DMEM with 1% FBS and either simvastatin (10 μM final concentration) or an equal volume of DMSO vehicle (control) for 24 h prior to harvest for measurement of the protein synthesis rate or eIF2B activity. For simvastatin treatment, preliminary experiments revealed maximal effects at 10 μM, intermediate effects at 1 μM, and no significant effect at 0.1 μM (Fig. S1; supplementary materials are found with the online version of the paper at the Journal website). Similar results were obtained for both myoblasts and differentiated myotubes.

Protein synthesis.

Cells were seeded and treated as described above. During the last 30 min of the 24-h simvastatin treatment, 5 μl of [³⁵S]EasyTag Express protein labeling mix (11.0 mCi/ml; NEN, Boston, MA) was added directly to the cell culture medium. Global rates of protein synthesis were estimated by the incorporation of [³⁵S]methionine and [³⁵S]cysteine into TCA-precipitable protein as previously described (9).

eIF2B activity assay.

The eIF2B guanine nucleotide exchange activity of control or simvastatin-treated cell lysates was assayed as previously described (9). Linear regression analysis was performed to calculate the slope for each assay, which was corrected for total protein content of the lysates as determined by detergent-compatible protein assay (Bio-Rad, Hercules, CA).

Western blot analyses.

For protein abundance and phosphorylation status, 2.0 × 10⁵ cells (for differentiation into myotubes and control myoblasts) or 2.5 × 10⁵ (for myoblasts treated with simvastatin) were seeded into six-well culture dishes and treated as described above. In some experiments, MG-132 (final concentration 25 μM) or an equal volume of DMSO vehicle was added directly to the medium for the final 8 h of simvastatin or DMSO treatment (i.e., hours 16–24). At the time of harvest, cells were washed with ice-cold PBS, harvested directly into 1× Laemmli sample buffer (200 μl), and boiled for 5 min at 100°C. An equal volume of sample was loaded into each lane of a Bio-Rad Criterion precast (4–15%) gel and subjected to SDS-PAGE and subsequent Western blotting as previously described (18). Membranes were incubated overnight at 4°C with one of the following primary antibodies diluted in TBS-T: eIF2Bα (rabbit polyclonal generously provided by Glen N. Barber, University of Miami); eIF2Bβ, -γ, -δ, and -ϵ (mouse monoclonal); eIF2α [mouse monoclonal (32)]; eIF2Bϵ phospho-Ser⁵³⁵ and eIF2α phospho-Ser⁵¹ (rabbit polyclonal; Biosource-Invitrogen); Akt phospho-Ser⁴⁷³ and total (rabbit polyclonal; Cell Signaling Technology, Danvers, MA); GSK-3β total (rabbit polyclonal, Calbiochem-EMD Biosciences, La Jolla, CA); GSK-3β phospho-Ser⁹ (rabbit polyclonal, Cell Signaling); and GAPDH (rabbit polyclonal; Santa Cruz Biotechnology, Santa Cruz, CA). All proteins analyzed for total protein abundance were normalized to GAPDH density for the corresponding sample, while phosphorylation states were quantitated as a ratio of phosphospecific band density to the density for the corresponding total protein abundance.

RNA isolation and quantitative real-time PCR analysis.

Mouse C₂C₁₂ myoblasts were seeded and treated as described in the procedures for Western blot analyses. At the time of harvest, cells were washed with ice-cold PBS and total cellular RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's protocol. RNA pellets were resuspended in 20 μl of Ambion RNA Storage Solution, and RNA quantity and purity were assessed via NanoDrop ND-1000 spectrophotometer (Thermo Scientific, Wilmington, DE). RNA (1 μg) was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA). The resulting cDNA was diluted 1:16 in nuclease-free water, and 2 μl of the diluted cDNA was used in a 20-μl reaction using the QuantiTect SYBR Green (Qiagen, Valencia, CA) master mix according to the manufacturer's protocol with gene-specific primers. The quantitative real-time PCR (qRT-PCR) analysis was performed in triplicate on a StepOnePlus Real-Time PCR system (Applied Biosystems) with a relative standard curve for each primer set. The mRNA abundance for each of the eIF2B subunits was normalized to GAPDH mRNA abundance for the corresponding sample (mouse GAPDH forward primer 5′-GTTGTCTCCTGCGACTTCA-3′, reverse primer 5′-TGCTGTAGCCGTATTCATTG-3′). Samples were analyzed from three independent experiments, each performed in triplicate, and are expressed as a ratio of the control values (mean ± SE). The primers directed against the mouse eIF2B subunits were as follows: eIF2Bβ, forward 5′-TAATGAGCTGCTGGTGGAACTGGA-3′, reverse 5′-AGCGCACTCTGCAACAATAACGTG-3′; eIF2Bγ, forward 5′-TGGGACTCTACATGGAAGCCAACA-3′, reverse 5′-AGGATGAGCCAATGACAGAGTGCT-3′; eIF2Bϵ, forward 5′-AGTTCTAGTGGCCGATAGCTT-3′, reverse 5′-AGCAGCAAAAGACAAATGTTTCC-3′.

Statistical analyses.

Data were analyzed with Prism version 5 (GraphPad Software). Data between two groups (i.e., control and simvastatin-treated samples) were compared via unpaired t-tests. For mevalonate and proteasome inhibition experiments comparing multiple treatment groups, a one-way analysis of variance (ANOVA) was employed followed by Newman-Keuls post hoc multiple comparisons (when the ANOVA detected significant differences among treatments). A value of P < 0.05 was considered statistically significant.

RESULTS

Treatment of cells with simvastatin for 24 h resulted in a significant reduction in global rates of protein synthesis as measured by incorporation of [³⁵S]methionine and [³⁵S]cysteine into protein, with similar effects observed for myoblasts (−26.43 ± 5.33% vs. control, P < 0.001) and myotubes (−22.40 ± 4.91% vs. control, P < 0.001). The reduction in protein synthetic rate due to simvastatin treatment was prevented by coincubation with 100 μM mevalonate (i.e., the product of HMG-CoA reductase; Fig. 1).

Fig. 1.

Simvastatin treatment represses rates of protein synthesis. C₂C₁₂ myotubes were treated with vehicle (control) or 10 μM simvastatin for 24 h in the absence or presence of 100 μM mevalonate as indicated. During the final 30 min of treatment, cells were incubated with a mixture of [³⁵S]Met/Cys for the measurement of incorporation of the radiolabeled amino acids into TCA-precipitable proteins. Results represent means ± SE of 3 experiments, each performed in triplicate, and are expressed as %mean control values. Differences among treatment groups (one-way ANOVA, P < 0.001) were subsequently analyzed via Newman-Keuls multiple comparison tests. Different letters denote statistical differences among treatment groups (P < 0.05); treatment groups with similar letters are not statistically different.

As described in the introduction, altered expression of the guanine nucleotide exchange factor eIF2B, particularly of its catalytic ϵ-subunit, is one mechanism implicated in the dysregulation of protein synthesis in skeletal muscle. In the present investigation, simvastatin treatment of myoblasts and myotubes significantly decreased the relative protein abundance of eIF2Bϵ (Fig. 2). The reduction in eIF2Bϵ expression was a direct result of the inhibition of HMG-CoA reductase, as coincubation of simvastatin-treated cells with 100 μM mevalonate prevented the decrease in eIF2Bϵ expression (Fig. 2). To examine the possibility that decreased eIF2Bϵ protein expression was a consequence of reduced mRNA abundance, qRT-PCR analysis was performed on control and simvastatin-treated myoblasts. The relative abundance of eIF2Bϵ mRNA was unaltered (1.00 ± 0.03 control vs. 1.02 ± 0.04 simvastatin treated). Another potential mechanism for reduced expression of eIF2Bϵ relative to other proteins in the simvastatin-treated cells is via degradation by the 26S proteasome. Several groups have implicated induction of components of the ubiquitin-proteasome system in skeletal muscle with statin use (11, 21, 36), which can lead to selective degradation of ubiquitinated target proteins. To examine this possibility, cells were treated with the proteasome inhibitor MG-132 during the final 8 h of simvastatin treatment. Inhibition of the proteasome during simvastatin treatment restored eIF2Bϵ protein expression to control values (Fig. 3, A and F). Based on the decrease in total eIF2Bϵ protein expression in response to simvastatin treatment, the effect of the drug on mRNA and protein expression of the other subunits of the holoenzyme was also assessed. Simvastatin treatment resulted in significantly reduced protein expression of the other four subunits of eIF2B following 24 h of treatment (Fig. 3, A–E). As with eIF2Bϵ, the presence of proteasome inhibitor for the final 8 h prevented the simvastatin-induced decrease of the four remaining subunits (α, β, γ, δ). The relative mRNA abundance for eIF2Bβ was unaffected by simvastatin treatment (1.00 ± 0.02 control vs. 0.97 ± 0.02 simvastatin treated); in contrast, the eIF2Bγ mRNA exhibited a small but significant reduction (1.00 ± 0.05 control vs. 0.81 ± 0.06 simvastatin treated; P < 0.05).

Fig. 2.

Simvastatin treatment reduces eukaryotic initiation factor (eIF)2Bϵ protein expression. C₂C₁₂ myoblasts (A) or myotubes (B) were treated with vehicle (control) or 10 μM simvastatin for 24 h in the absence or presence of 100 μM mevalonate as indicated. Equal volumes of cell lysates were subjected to Western blot analysis using antibodies against eIF2Bϵ and normalized to GAPDH abundance for the corresponding sample. Results in each panel represent means ± SE of 3 experiments, each performed in triplicate, and are expressed as %mean control values. Differences among treatment groups (one-way ANOVA, P < 0.05) were subsequently analyzed via Newman-Keuls multiple comparison tests. Different letters denote statistical differences among treatment groups (P < 0.05); treatment groups with similar letters are not statistically different.

Fig. 3.

Simvastatin treatment reduces eIF2B subunit protein expression in a proteasome-sensitive manner. C₂C₁₂ myoblasts were treated with vehicle (control) or 10 μM simvastatin for 24 h. MG-132 (25 μM) or vehicle was added to subsets of cells for the final 8 h of incubation as indicated. Equal volumes of cell lysates were subjected to Western blot analysis using antibodies against eIF2B subunits. Protein abundance for each subunit was normalized to GAPDH abundance for the corresponding sample. A: representative Western blots for each of the 5 subunits and GAPDH. B–F: bar graphs representing quantitation (means ± SE) of 3–4 independent experiments each performed in triplicate for eIF2Bα (B), eIF2Bβ (C), eIF2Bγ (D), eIF2Bδ (E), and eIF2Bϵ (F). Differences among treatment groups occurred for all 5 subunits (one-way ANOVA, P < 0.0001) and were subsequently analyzed via Newman-Keuls multiple comparison tests. Different letters denote statistical differences among treatment groups (P < 0.05); treatment groups with similar letters are not statistically different.

Since eIF2B activity is a rate-controlling step in translation initiation and has been implicated in the control of protein synthesis in skeletal muscle (7, 19) and myoblasts (16), we measured the GEF activity of eIF2B by use of an in vitro assay. The activity of eIF2B in the lysates of cells treated with simvastatin was repressed by ∼20% compared with control cells (Fig. 4A). To examine additional potential mechanisms that might account for repression of eIF2B activity, Western blots were performed to assess the phosphorylation of Akt (Ser⁴⁷³), GSK-3β (Ser⁹), eIF2α (Ser⁵¹) and eIF2Bϵ (Ser⁵³⁵). Simvastatin treatment resulted in decreased Akt phosphorylation at Ser⁴⁷³ (Fig. 4B), with concomitant reductions in GSK-3β phosphorylation at Ser⁹ (Fig. 4C). Reduced phosphorylation of GSK-3β at Ser⁹ leads to increased activity of the kinase toward its substrates, including eIF2Bϵ. In accord with the changes in Akt and GSK-3β phosphorylation, the relative phosphorylation of eIF2Bϵ at Ser⁵³⁵ was significantly increased in response to statin treatment (Fig. 4D). Phosphorylation of eIF2Bϵ at Ser⁵³⁵ is considered to be repressive with regard to the enzyme's activity (14, 41), which is consistent with the observed results. It is important to note that the absolute amount of eIF2Bϵ present in the phosphorylated form was unchanged in response to simvastatin treatment. However, because total eIF2Bϵ abundance was reduced, the relative phosphorylation was increased. Thus, the change in relative phosphorylation may not necessarily reflect a change in GSK-3 activity toward eIF2Bϵ. Another well-known mechanism resulting in inhibition of eIF2B activity is phosphorylation of the α-subunit of its substrate (eIF2α) at Ser⁵¹ (30). In the experiments performed herein, simvastatin treatment did not alter eIF2α phosphorylation at Ser⁵¹ (Fig. 4E), suggesting that phosphorylation of eIF2α was not a factor contributing toward the observed reduction in eIF2B activity.

Fig. 4.

Repression of eIF2B guanine nucleotide exchange activity in response to simvastatin treatment is associated with altered phosphorylation of Akt, GSK-3β, and eIF2Bϵ. C₂C₁₂ myoblasts were treated with vehicle (control) or 10 μM simvastatin for 24 h. A:lysates of control and simvastatin-treated myoblasts were subjected to eIF2B guanine nucleotide exchange activity assay. Results represent means ± SE of 3 experiments, each performed at least in triplicate, and are expressed as %mean control values. *P < 0.05 vs. control. B–F: equal volumes of cell lysates were subjected to Western blot analysis using antibodies specific for proteins phosphorylated at a specific site or for the total protein. Phosphorylation of Akt on Ser⁴⁷³ (B), GSK-3β on Ser⁹ (C), eIF2Bϵ on Ser⁵³⁵ (D), and eIF2α on Ser⁵¹ (E) were normalized to the respective total protein and expressed as %mean control values. Representative blots are shown in each panel with control and simvastatin-treated samples in triplicates. Results represent quantitation (means ± SE) of 3 independent experiments with 3 replicates per experiment. * P < 0.05 vs. control.

DISCUSSION

The mechanisms behind statin-associated myopathies remain perplexing, and a number of possibilities have been proposed and/or implicated, including mitochondrial toxicity and induction of apoptosis (37), autophagy (1), enhanced proteolytic degradation via multiple pathways (21), and repressed protein synthesis (8, 22, 28). In the present investigation, we show that simvastatin treatment of the muscle-derived C₂C₁₂ cell line results in a repression of global rates of protein synthesis, in part via alteration in eIF2B expression and guanine nucleotide exchange activity. The reduction in eIF2B activity was associated with increased relative phosphorylation of eIF2Bϵ as well as a proteasome-sensitive reduction in eIF2B subunit protein expression. The effect of MG-132 in statin-treated cells implies that eIF2B may be targeted for degradation by the proteasome during simvastatin treatment. Little is known regarding the stability of the eIF2B complex in mammalian cells; however, a single mutation in any one of several of the eIF2B subunits can lead to decreased protein stability of the subunit as well as the stability of other subunits (29). In the current investigation, it is possible that one subunit may have been ubiquitinated and targeted to the proteasome, whereupon the remaining subunits of the complex become more labile, resulting in reduced expression of the additional subunits (which can be prevented by proteasome inhibition). An alternative explanation is an indirect effect whereby the proteasome targets interacting or upstream regulatory protein(s) that affects eIF2B subunit expression.

The direct mechanism of action of the statin family of drugs is the inhibition of HMG-CoA reductase resulting in reduced cholesterol biosynthesis; however, an additional effect is reduced production of the isoprenoid intermediates of the cholesterol biosynthetic pathway. By reducing substrate availability, proteins that are normally isoprenylated (i.e., modified with farnesyl or geranyl-geranyl moieties) are affected by the statins, and their function and/or membrane localization may be impaired (19). Particularly susceptible to such effects are the small GTPases (e.g., Ras, Rac, Rho). With regard to protein synthesis, impaired farnesylation of the Rheb GTPase (an upstream activator of the mTORC1 complex), has been demonstrated in response to atorvastatin (7) and lovastatin treatment (39) in different cell types. Our laboratory has reported that mTORC1 activation is necessary for stimulation of eIF2Bϵ subunit protein synthesis (18); in light of reduced Akt phosphorylation as well as impaired farnesylation of Rheb with statin treatment, the synthesis of eIF2Bϵ may be repressed. However, repressed translation of the eIF2Bϵ mRNA is unlikely to explain the results of the present investigation on the basis of the ability of MG-132 to prevent the decrease in eIF2Bϵ protein expression as well as several other subunits of eIF2B during simvastatin treatment. While none of the eIF2B subunits possesses the canonical carboxyl-terminal CaaX isoprenylation motif, at least one subunit (eIF2Bα) has been reported to colocalize with adrenergic receptors along the plasma membrane (17). Consequently, one possibility is that an interacting protein of one or more of the eIF2B subunits is normally isoprenylated and that simvastatin, by impairing this modification, alters the normal stability (i.e., synthesis and/or degradation) of eIF2B subunits and/or the activity of the eIF2B holoenzyme, perhaps by altering cellular localization. It is also important to consider that some of the effects of statins occur in an HMG-CoA reductase inhibition-independent manner. For example, IGF-I signaling is impaired in differentiating C₂C₁₂ myoblasts treated with simvastatin, but the effects are not rescued by addition of mevalonate (the product of HMG-CoA reductase) or its isoprenoid derivatives (27). However, in the present investigation, the simvastatin-induced repression of protein synthesis and eIF2Bϵ protein expression were both completely prevented by the addition of mevalonate, implicating the product of HMG-CoA reductase as a mediator of these effects.

Although treatment with MG-132 was able to rescue eIF2B subunit expression, we did not measure eIF2B activity or protein synthesis in such conditions, because treatment with MG-132 has been shown to increase eIF2α (Ser⁵¹) phosphorylation and reduce protein synthesis rates in several cell types including C₂C₁₂ myoblasts (6, 15, 44). Indeed, we observed an approximately threefold increase in eIF2α phosphorylation at Ser⁵¹ after incubation with MG-132 (data not shown). Thus, preventing the loss of eIF2B subunit expression via proteasome inhibition would not be expected to rescue the reduction in eIF2B activity or protein synthesis rates due to the concomitant increase in eIF2α phosphorylation that would directly inhibit eIF2B activity.

One important regulator in common to both the protein synthesis and degradation pathways is the AGC serine-threonine kinase Akt. Decreased phosphorylation of Akt has been reported in response to statin treatment in the mouse C₂C₁₂ myoblast cell line (11, 27) as well as in rat skeletal muscle in vivo (21). One downstream consequence of reduced Akt activity in response to statins is increased expression of the E3 ligases MAFbx and MuRF1 that are associated with muscle protein degradation (11, 21). However, another consequence to a reduction in Akt activity is enhanced activity of the Akt substrate GSK-3β. Reduced phosphorylation and thus increased activity of GSK-3β can lead to increased phosphorylation of eIF2B at an inhibitory site of its catalytic ϵ-subunit (14, 41). In the present investigation, we confirmed the reduction in Akt Ser⁴⁷³ phosphorylation and observed a significant reduction in GSK-3β phosphorylation at Ser⁹. Although we cannot directly implicate GSK-3β activity in the phosphorylation of eIF2Bϵ in the conditions used in the present investigation, the reduction in total eIF2Bϵ expression resulted in a relative increase in eIF2Bϵ phosphorylation at Ser⁵³⁵. Finally, reduced Akt phosphorylation at Ser⁴⁷³ would also be expected to reduce the activity of the mTORC1 complex, another critical effector of the control of protein synthesis. The effect of statins on reducing mTORC1 signaling has been reported by others in a number of different cell types (7, 31, 39). Accordingly, the results of the present study regarding eIF2B expression represent only one potential effector of reduced protein synthesis rates.

Another potential limitation of the present investigation is that the concentration of simvastatin used was 10 μM; this dose was chosen on the basis of preliminary experiments as well as an abundance of published investigations in which similar or higher concentrations of statins were used in cell culture experiments, including C₂C₁₂ cells. The reported concentration of statins in the plasma of human patients with normal therapeutic doses is usually submicromolar. However, in some patients who experience statin-related myopathies (e.g., due to drug interactions, polymorphisms in organic anion-transporting polypeptides, etc.), statin concentrations may indeed reach the low micromolar range (25, 33). Importantly, we have observed the reduction in eIF2Bϵ protein expression with 10 μM but not 0.1 μM simvastatin treatment for 24 h, whereas 1 μM resulted in an intermediate effect (Supplementary Fig. S1). Nevertheless, the implication of the dose-related effect of simvastatin on eIF2B that we observed in cell culture in the context of skeletal muscle of human patients remains unclear.

In summary, the results of the present study implicate reduced eIF2B holoenzyme expression in the reduction of protein synthesis during statin treatment in C₂C₁₂ myoblasts. Repression of eIF2B guanine nucleotide exchange activity is a well-known mechanism for the regulation of global rates of protein synthesis. The reduction in eIF2B activity appears to be primarily the result of decreased eIF2B subunit abundance, with a resulting relative increase in eIF2Bϵ phosphorylation at the inhibitory Ser⁵³⁵ residue of eIF2Bϵ. The precise mechanism for the reduction in eIF2B subunit expression is not known but is dependent on the ubiquitin-proteasome system. Future studies directed at further examining the mechanism(s) of reduced protein synthesis and its effects on statin-associated myopathies will help in understanding the complexity of this class of beneficial pharmacological agents.

GRANTS

The studies described here were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-15658 (to L. S. Jefferson) and a grant from The Pennsylvania Department of Health using Tobacco Settlement Funds.

The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions.

DISCLOSURES

No conflicts of interest are reported by the authors.