Simvastatin Downregulates Cofilin and Stathmin to Inhibit Skeletal Muscle Cells Migration

Statins are the most effective therapeutic agents for reducing cholesterol synthesis. Given their widespread use, many adverse effects from statins have been reported; of these, musculoskeletal complications occurred in 15% of patients after receiving statins for 6 months, and simvastatin was the most commonly administered statin among these cases. This study investigated the negative effects of simvastatin on skeletal muscle cells. We performed RNA sequencing analysis to determine gene expression in simvastatin-treated cells. Cell proliferation and migration were examined through cell cycle analysis and the transwell filter migration assay, respectively. Cytoskeleton rearrangement was examined through F-actin and tubulin staining. Western blot analysis was performed to determine the expression of cell cycle-regulated and cytoskeleton-related proteins. Transfection of small interfering RNAs (siRNAs) was performed to validate the role of cofilin and stathmin in the simvastatin-mediated inhibition of cell migration. The results revealed that simvastatin inhibited the proliferation and migration of skeletal muscle cells and affected the rearrangement of F-actin and tubulin. Simvastatin reduced the expression of cofilin and stathmin. The knockdown of both cofilin and stathmin by specific siRNA synergistically impaired cell migration. In conclusion, our results indicated that simvastatin inhibited skeletal muscle cell migration by reducing the expressions of cofilin and stathmin.


Introduction
Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and the most effective therapeutic agents used to reduce cholesterol synthesis and the low-density lipoprotein level in blood [1][2][3]. Statins are used for the treatment and prevention of hyperlipidemia and can reduce the incidence of cardiovascular events in patients. However, as the use of statins has become widespread, many adverse effects have been reported, especially statin-associated myopathy, such as muscle cramping, soreness, fatigue, weakness, myositis, and rhabdomyolysis. A study reported that 10-15% of patients developed musculoskeletal complications after receiving statins for 6 months [4]. The side effects of statins often become apparent during or after strenuous bouts of exercise [5,6]. Among statin-associated myopathy, muscle weakness was observed in 58% of patients receiving statins, and simvastatin was the most commonly administered statin among these patients (49% of patients) [7]. The FDA Adverse Event Reporting System database reported that 36% of statin-associated rhabdomyolysis was associated with simvastatin from November 1997 through March 2000 [8].
It has been reported that statins impair skeletal muscle healing on postinjury [9,10]. Muscle healing is a complex process that involves inflammatory, repair, and remodeling phases. In the inflammatory phase, neutrophils and macrophages invade the injured site

Simvastatin Moderated the Expression of Genes Related to Cell Proliferation and Migration in Skeletal Muscle Cells
To investigate the effect of simvastatin on skeletal muscle cells, we performed RNAsequencing to analyze gene expression in skeletal muscle cells treated with 10 µM simvastatin. A total of 2355 differentially expressed genes (DEGs) were identified between control group and 10 µM simvastatin group. Simvastatin upregulated and downregulated the expression of 968 and 1387 genes, respectively, compared with control (Supplementary Figure S1). The results of Gene ontology (GO)enrichment analysis revealed that DEGs downregulated by simvastatin were related to nuclear division, DNA replication, and microtubule cytoskeleton organization (Figure 1). The results indicated that simvastatin inhibited the expression of genes related to cell proliferation and migration.

Simvastatin Affected Cell Proliferation by Downregulating Cyclin E, Cyclin A, and Cdk1
The findings of RNAsequencing analysis indicated that simvastatin inhibited the expression of cell cycle regulation genes, namely cyclin E, cyclin A, and Cdk1. Furthermore, we investigated the effect of simvastatin on cell viability and the cell cycle. Skeletal muscle cells were treated with varying concentrations of simvastatin for 24 h; the MTT assay was performed to examine the viability of skeletal muscle cells. The results revealed that the viability of skeletal muscle cells decreased after simvastatin treatment. The relative cell viability was 100.0% ± 4.0% in control cells and 97.2% ± 5.1%, 95.1% ± 3.9%, and 78.0% ± 3.7% in cells treated with 1, 5, and 10 μM simvastatin, respectively ( Figure 2a). The results of cell cycle analysis indicated that simvastatin caused G1 phase arrest and inhibited the S and G2 phases of skeletal muscle cells (Figure 2b). We observed that 74.6% ± 3.3% of control cells were in the G1 phase and 76.0% ± 2.8%, 83.2% ± 2.7%, and 85.5% ± 1.0% of skeletal muscle cells treated with 1, 5, and 10 μM simvastatin were in the G1 phase, respectively. Furthermore, 12.3% ± 2.0% of control cells were in the S phase, and 12.2% ± 1.3%, 6.5% ± 1.7%, and 5.5% ± 0.2% of skeletal muscle cells treated with 1, 5, and 10 μM simvastatin were in the S phase, respectively. We noted that 13.1% ± 1.4% of control cells were in the G2 phase and 11.8% ± 1.5%, 10.3% ± 1.0%, and 9.0% ± 0.8% of skeletal muscle cells treated with 1, 5, and 10 μM simvastatin were in the G1 phase, respectively. The protein expression of cyclin E, cyclin A, and CDK1 was determined through Western blot analysis ( Figure 2c); the band intensity is shown in Figure 2d. The results revealed that simvastatin suppressed the protein expression of cylin E, cyclin A, and CDK1.

Simvastatin Affected Cell Proliferation by Downregulating Cyclin E, Cyclin A, and Cdk1
The findings of RNAsequencing analysis indicated that simvastatin inhibited the expression of cell cycle regulation genes, namely cyclin E, cyclin A, and Cdk1. Furthermore, we investigated the effect of simvastatin on cell viability and the cell cycle. Skeletal muscle cells were treated with varying concentrations of simvastatin for 24 h; the MTT assay was performed to examine the viability of skeletal muscle cells. The results revealed that the viability of skeletal muscle cells decreased after simvastatin treatment. The relative cell viability was 100.0% ± 4.0% in control cells and 97.2% ± 5.1%, 95.1% ± 3.9%, and 78.0% ± 3.7% in cells treated with 1, 5, and 10 µM simvastatin, respectively ( Figure 2a). The results of cell cycle analysis indicated that simvastatin caused G1 phase arrest and inhibited the S and G2 phases of skeletal muscle cells (Figure 2b). We observed that 74.6% ± 3.3% of control cells were in the G1 phase and 76.0% ± 2.8%, 83.2% ± 2.7%, and 85.5% ± 1.0% of skeletal muscle cells treated with 1, 5, and 10 µM simvastatin were in the G1 phase, respectively. Furthermore, 12.3% ± 2.0% of control cells were in the S phase, and 12.2% ± 1.3%, 6.5% ± 1.7%, and 5.5% ± 0.2% of skeletal muscle cells treated with 1, 5, and 10 µM simvastatin were in the S phase, respectively. We noted that 13.1% ± 1.4% of control cells were in the G2 phase and 11.8% ± 1.5%, 10.3% ± 1.0%, and 9.0% ± 0.8% of skeletal muscle cells treated with 1, 5, and 10 µM simvastatin were in the G1 phase, respectively. The protein expression of cyclin E, cyclin A, and CDK1 was determined through Western blot analysis ( Figure 2c); the band intensity is shown in Figure 2d. The results revealed that simvastatin suppressed the protein expression of cylin E, cyclin A, and CDK1.

Simvastatin Reduced the Migration Ability of Skeletal Muscle Cells
As shown in Figure 2, simavastatin inhibited the proliferation of skeletal muscle cells. We next performed the transwell filter migration assay to evaluate the migration ability of skeletal muscle cells treated with simvastatin for 24 h. The results revealed that simvastatin reduced the migration ability of skeletal muscle cells (Figure 3a). The relative migration rate was 100.0% ± 3.6% in control cells and 92.4% ± 5.5%, 19.6% ± 2.2%, and 7.7% ± 1.

Simvastatin Reduced the Migration Ability of Skeletal Muscle Cells
As shown in Figure 2, simavastatin inhibited the proliferation of skeletal muscle cells. We next performed the transwell filter migration assay to evaluate the migration ability of skeletal muscle cells treated with simvastatin for 24 h. The results revealed that simvastatin reduced the migration ability of skeletal muscle cells (Figure 3a). The relative migration rate was 100.0% ± 3.6% in control cells and 92.4% ± 5.5%, 19.6% ± 2.2%, and 7.7% ± 1.1% in cells treated with 1, 5, and 10 μM simvastatin ( Figure 3b). The differences between the groups were statistically significant (p < 0.05).  Results are presented as the mean ± standard error of the mean of six independent experiments. * mean p < 0.05 compared with control. Scale bars: 60 μm.

Simvastatin Inhibited the Spreading of Skeletal Muscle Cells
As shown in Figure 3, simvastatin reduced the migration ability of skeletal muscle cells. The initial step of cell migration is cell attachment and spreading. We examined the cell spreading of skeletal muscle cells after simvastatin treatment (Figure 4a). The numbers of spreading and adhesive cells were separately counted at 30, 60, and 90 min after plating. At 90 min, the relative percentage of spreading was 87.9% ± 2.1% for control

Simvastatin Inhibited the Spreading of Skeletal Muscle Cells
As shown in Figure 3, simvastatin reduced the migration ability of skeletal muscle cells. The initial step of cell migration is cell attachment and spreading. We examined the cell spreading of skeletal muscle cells after simvastatin treatment ( Figure 4a). The numbers of spreading and adhesive cells were separately counted at 30, 60, and 90 min after plating. At 90 min, the relative percentage of spreading was 87.9% ± 2.1% for control cells and 80.4% ± 4.2%, 58.5% ± 7.7%, and 42.8% ± 7.8% for cells treated with 1, 5, and 10 µM simvastatin, respectively (p < 0.05; Figure 4b). In addition, the F-actin distribution range of spreading cells was wider than that of nonspreading cells. The results indicated that simvastatin reduced cell spreading characterized by the appearance of pseudopodia extensions (Figure 4c).

Effect of Simvastatin on the Cytoskeleton Distribution of Skeletal Muscle Cells
During the cell migration process, migrating cells are driven by cytoskeleton-associated proteins, namely F-actin and tubulin. We performed immunofluorescence staining to observe F-actin and tubulin in skeletal muscle cells treated with simvastatin. The distributions of F-actin and tubulin in skeletal muscle cells both changed after simvastatin treatment. The expression of F-actin was decreased after simvastatin treat-

Effect of Simvastatin on the Cytoskeleton Distribution of Skeletal Muscle Cells
During the cell migration process, migrating cells are driven by cytoskeleton-associated proteins, namely F-actin and tubulin. We performed immunofluorescence staining to observe F-actin and tubulin in skeletal muscle cells treated with simvastatin. The distributions of F-actin and tubulin in skeletal muscle cells both changed after simvastatin treatment. The expression of F-actin was decreased after simvastatin treatment and was significantly decreased in cells treated with 10 µM simvastatin (Figure 5a). However, the formation of tubulin exhibited differences after simvastatin treatment. Tubulin became longer and bundled in the high-dose group (5 and 10 µM; Figure 5b). The results indicated that simvastatin changed the organization of F-actin and tubulin.
22, x FOR PEER REVIEW 7 of 15 ment and was significantly decreased in cells treated with 10 μM simvastatin ( Figure  5a). However, the formation of tubulin exhibited differences after simvastatin treatment.
Tubulin became longer and bundled in the high-dose group (5 and 10 μM; Figure 5b). The results indicated that simvastatin changed the organization of F-actin and tubulin. Tubulin was stained red, and nuclei were stained blue. The photographs were obtained using a fluorescence microscope (200×). Scale bars: 60 μm.

Simvastatin Reduced the Expression of Cytoskeleton-Associated Proteins in Skeletal Muscle Cells
Skeletal muscle cells were untreated or treated with 1, 5, or 10 μM simvastatin; the protein extracts were analyzed through Western blot analysis. The protein expressions of stathmin and cofilin are shown in Figure 6a. The band intensity of cofilin was 100.0% ± 4.6% in control cells and 60.0% ± 2.3%, 40.6% ± 1.6%, and 28.8% ± 1.1% in cells treated with 1, 5, and 10 μM simvastatin, respectively. The band intensity of stathmin was 100.0% ± 3.2% in control cells and 88.2% ± 2.1%, 46.4% ± 1.5%, and 20.5% ± 0.6% in cells treated with 1, 5, and 10 μM simvastatin, respectively (Figure 6b). The results of Western blot and band intensity analyses indicated that simvastatin suppressed the protein expression of cofilin and stathmin.

Simvastatin Reduced the Expression of Cytoskeleton-Associated Proteins in Skeletal Muscle Cells
Skeletal muscle cells were untreated or treated with 1, 5, or 10 µM simvastatin; the protein extracts were analyzed through Western blot analysis. The protein expressions of stathmin and cofilin are shown in Figure 6a. The band intensity of cofilin was 100.0% ± 4.6% in control cells and 60.0% ± 2.3%, 40.6% ± 1.6%, and 28.8% ± 1.1% in cells treated with 1, 5, and 10 µM simvastatin, respectively. The band intensity of stathmin was 100.0% ± 3.2% in control cells and 88.2% ± 2.1%, 46.4% ± 1.5%, and 20.5% ± 0.6% in cells treated with 1, 5, and 10 µM simvastatin, respectively (Figure 6b). The results of Western blot and band intensity analyses indicated that simvastatin suppressed the protein expression of cofilin and stathmin.

Discussion
The potential mechanisms of statin-induced myopathy include reduced levels of isoprenoids, mitochondria dysfunction, and genetic factor. The isoprenoids are intermediates of the mevalonate pathway. Statin inhibited HMG-CoA reductase in cholesterol biosynthesis and reduced isoprenoid compounds. The isoprenoids, such as geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate, dolichols and ubiquinone (coenzyme Q10), are involved in posttranslational modifications of small GTPases, protein glycosylation, mitochondria biogenesis, which is essential for cell growth, gene expression, and cytoskeleton assembly [4,22]. Most of the small GTPases, such as the Rho and Ras families, are related to cell behaviors, including cell proliferation, cell migration, and apoptosis. Several studies have reported that simvastatin inhibited the proliferation and migration of cancer cells [23][24][25][26]. The proliferation of skeletal muscle cells is regulated by the eukaryotic cell cycle [13]. Similar to other types of cells, muscle cells have four cell division cycle stages: G1, S, G2, and M. The chromosomal DNA of cells replicates during the S phase. When the cell cycle moves from the S to G2-M stages, cells begin to divide. The cell cycle is accurately controlled by cyclins and Cdk complexes [14]. Our previous study indicated that simvastatin suppressed tendon cell proliferation. Simvastatin reduced the expression of cyclins and cdks and altered cell cycle progression. We observed that the supplementation of GGPP reversed the inhibition of cell proliferation in tendon cells [27]. In this study, simvastatin reduced the viability of skeletal muscle cells. The GO enrichment analysis indicated that DEGs downregulated by simvastatin were related to nuclear division, chromosome organization, DNA replication, and regulation of mitotic cell cycle. Moreover, simvastatin inhibited skeletal muscle cell proliferation and caused cell cycle arrest at the G1/S transition. The underlying molecular mechanism appears to be related to the downregulation of cyclin E, cyclin A, and Cdk1.
The GO enrichment analysis also revealed that DEGs downregulated by simvastatin were related to microtubule cytoskeleton organization. A Study indicated that simvastatin inhibits the migration and adhesion of monocytes and disorganizes F-actin in endothelial cells [28]. In this study, simvastatin reduced the migration and spreading of skeletal muscle cells. F-actin organization was inhibited during the spreading process ( Figure 2). We performed immunofluorescence staining to examine the cytoskeleton organization after simvastatin treatment, including that of actin microfilaments (F-actin) and microtubules (tubulin). F-actin was decreased; tubulin clustered together after simvastatin treatment. The results indicated that simvastatin likely affected the cytoskeleton organization of skeletal muscle cells. The results of this study revealed that simvastatin downregulated the expression of cofilin and stathmin. Cofilin depolymerizes F-actin to G-actin by binding to the ADP-bound actin of F-actin. The activity of cofilin was regulated by the phosphorylation and dephosphorylation of serine 3 residues [29]. Several studies have indicated that the defect in cofilin inhibited cell spreading and migration in cancer metastasis [30,31]. Furthermore, a previous study indicated that simvastatin reduced the expression of Factin and attenuated neuropathic pain by inhibiting the RhoA/LIMK/cofilin pathway in a chronic neuropathic pain model [32]. Stathmin is a key regulator that depolymerizes microtubules. Phosphorylation at Ser 16 and Ser 38 inactivates stathmin and stabilizes microtubules; the phosphorylation of stathmin is regulated by CDKs. Moreover, the signal transducer and activator of transcription was associated with the activity of stathmin as an antagonist in microtubule polymerization [33]. Stathmin was observed to be frequently overexpressed in breast cancer cells and to mediate the resistance of antimicrotubule agents that are used as chemotherapy drugs [34]. However, the knockdown of stathmin expression reduced cell migration in esophageal squamous cell carcinoma cells and neuroblastoma cells [35,36]. Stathmin is involved in keratinocyte migration during cutaneous regeneration [37]. We used specific siRNAs to knockdown the expression of cofilin and stathmin. The cell migration rates of the cofilin and stathmin siRNA groups were lower than that of the control siRNA group. However, the cell migration rate of the cofilin plus stathmin siRNA group was lower than that of the single siRNA group. The results indicated that the lack of cofilin and stathmin in skeletal muscle cells inhibited cell migration ability. In conclusion, simvastatin inhibited the proliferation of skeletal muscle cells by downregulation of cyclin E, cyclin A, and Cdk1, and inhibited migration of skeletal muscle cells by downregulating cofilin and stathmin. This might be a potential mechanism of statin-induced myopathy that exerts a negative effect on muscle healing.
The results of this study revealed that simvastatin downregulated the expression of cofilin and stathmin. Cofilin depolymerizes F-actin to G-actin by binding to the ADP-bound actin of F-actin. The activity of cofilin was regulated by the phosphorylation and dephosphorylation of serine 3 residues [27]. Several studies have indicated that the defect in cofilin inhibited cell spreading and migration in cancer metastasis [28,29]. Furthermore, a previous study indicated that simvastatin reduced the expression of F-actin and attenuated neuropathic pain by inhibiting the RhoA/LIMK/cofilin pathway in a chronic neuropathic pain model [30]. Stathmin is a key regulator that depolymerizes microtubules. Phosphorylation at Ser 16 and Ser 38 inactivates stathmin and stabilizes microtubules; the phosphorylation of stathmin is regulated by CDKs. Moreover, the signal transducer and activator of transcription was associated with the activity of stathmin as an antagonist in microtubule polymerization [31]. Stathmin was observed to be frequently overexpressed in breast cancer cells and to mediate the resistance of antimicrotubule agents that are used as chemotherapy drugs [32]. However, the knockdown of stathmin expression reduced cell migration in esophageal squamous cell carcinoma cells and neuroblastoma cells [33,34]. Stathmin is involved in keratinocyte migration during cutaneous regeneration [35]. We used specific siRNAs to knockdown the expression of cofilin and stathmin. The cell migration rates of the cofilin and stathmin siRNA groups were lower than that of the control siRNA group. However, the cell migration rate of the cofilin plus stathmin siRNA group was lower than that of the single siRNA group. The results indicated that the lack of cofilin and stathmin in skeletal muscle cells inhibited cell migration ability. In conclusion, simvastatin inhibited the spreading and migration of skeletal muscle cells by downregulating cofilin and stathmin and might exert a negative effect on muscle healing.

Animals
Sprague-Dawley (SD) rats (male, 250 g) were obtained from BioLASCO Taiwan Co., Ltd. Taipei, Taiwan. All animal experimental procedures were approved by the Institutional Animal Care and Use Committee of Chang Gung University (CGU16-026).

Primary Culture of Rat Skeletal Muscle Cells
Skeletal muscle cells were isolated from the gastrocnemius muscle of SD rats. The isolation method was described previously [38,39]. The gastrocnemius muscle was treated with 2 mg/mL of type I collagenase (Sigma-Aldrich, St. Louis, MO, USA) for 1 h at 37 • C in a humidified incubator (5% CO 2 /95% air), followed by treatment with 0.25% trypsin-EDTA for 1 h. The supernatant was filtered using a 70-µm cell strainer. After centrifugation (1500 rpm, 5 min), cell pellets were suspended in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS) and 5% chick embryo extract (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and were seeded on a culture plate. After 1 h, the supernatant containing skeletal muscle cells was transferred to another culture plate and incubated at 37 • C in a humidified incubator (5% CO 2 /95% air). These cells were used in the subsequent experiments.

RNA Sequencing Analysis
Skeletal muscle cells were untreated or treated with 10 µM simvastatin (Sigma-Aldrich, St. Louis, MO, USA) for 24 h; total RNA was extracted using Trizol (Thermo Fisher Scientific, Waltham, MA, USA). RNA sequencing analysis was performed using Illumina NovaSeq 6000 (Illumina, Inc., San Diego, CA, USA). Differential expression analysis between the control group and the simvastatin-treated group was performed using DESeq2 (version 1.16.0., available at http://www.bioconductor.org/packages/release/bioc/html/DESeq2.html. Accessed dates: 27 October 2021). An adjusted p value of <0.05 and a |log2fold change| of >1 served as cutoff criteria for the selection of differentially expressed genes (DEGs). Gene ontology (GO) enrichment was performed using DEGs (adjusted p value ≤ 0.05).

Cells Spreading Assay and F-Actin Staining
Skeletal muscle cells treated with or without simvastatin for 24 h were subcultured and plated on six-well culture plates. After plating for 30, 60, and 90 min, the cells were observed and photographed under a light microscope (100×; CKX53; OLYMPUS, Tokyo, Japan). The cells with a round shape and bright appearance were considered to be nonspreading cells. The cells exhibiting pseudopodia extensions were considered to be spreading cells. The numbers of spreading and nonspreading cells were calculated in four random microscopic fields. For F-actin staining, the cells were fixed in 10% formalin for 15 min after plating for 30, 60 or 90 min. The cells were permeabilized using 0.1% Triton-X 100 in PBS for 5 min. After washing three times with PBS, the cells were incubated in blocking solution (3% BSA in PBS) at room temperature for 30 min and were incubated for 1 h with phalloidin-conjugated fluorescein isothiocyanate (FITC; Sigma, St. Louis, MO, USA) diluted in blocking solution. The cells were then washed in PBS and stained with PBS containing 1 µg/mL DAPI (Thermo Fisher Scientific, Waltham, MA, USA) for 5 min. Subsequently, the cells were washed in PBS and were examined using the ZOE Fluorescent Cell Imager (175×; Bio-Rad, Hercules, CA, USA).

Immunofluorescence Staining
Skeletal muscle cells were seeded on glass coverslips. The cells were untreated or treated with simvastatin for 24 h and fixed in 10% formalin for 15 min. After washing three times with PBS, glass coverslips were incubated in blocking solution (3% BSA in PBS) at room temperature for 30 min and incubated for 2 h with phalloidin-conjugated FITC (Sigma-Aldrich, St. Louis, MO, USA) or anti-tubulin antibody (1/200 dilution, Thermo Fisher Scientific, Waltham, MA, USA) or anti-stathmin antibody (Cell Signaling Technology, Danvers, MA, USA) diluted in blocking solution. The signal was detected using antirabbit IgG Alexa Fluor 488 or antimouse IgG Alexa Fluor 594 (Thermo Fisher Scientific, Waltham, MA, USA). After washing in PBS, the cells were stained with PBS containing 300 nM DAPI for 5 min. After washing in PBS, the cells were observed under a fluorescence microscope (200×; Eclipse Ni-U; Nikon, Tokyo, Japan).

Statistical Analysis
All data were expressed as the mean ± standard error of the mean. Comparisons between groups were performed using the Kruskal-Wallis test. The Mann-Whitney U test was used to identify differences between the groups. A p value of <0.05 indicated statistical significance.