Fig 1.
Model of atorvastatin-induced cytotoxicity in RD skeletal muscle cells.
(a) Determination of the IC50 for RD cells treated with atorvastatin (ATO) for 72h, indicating the concentration required to inhibit 50% of cell viability. (b) Electrophoretic mobility assay of RAP1 protein in RD cells treated with atorvastatin at 24 and 48 hours. (c) Targeted mass spectrometry analysis of ions in the m/z range of 372–379. The green peak represents cells cultured in media supplemented with 2-¹³C-acetate. The red peak corresponds to cells cultured with 2-¹³C-acetate and treated with atorvastatin (blue peak) for 24h, while the green peak indicates cells grown in standard media without atorvastatin at the same time. (d) Western blot (WB) analysis of biomarkers associated with energetic stress (AMPK, pT172-AMPK), cell differentiation (MyoD1, Myogenin), muscle stress (FBX32), protein unfolding or damage (BiP), DNA damage (p53, pSer20-p53), autophagy (p62, Lc3b), and apoptosis (PARP) in RD cells treated with atorvastatin (in IC50 fractions) for 72 h. As positive controls, cells were treated with amsacrine (10 µM) or DTT (0.5 mM) for 24 or 48 hours.
Fig 2.
Metabolomic analysis of RD cells treated with non-cytotoxic levels of atorvastatin.
Untargeted metabolomic analysis of RD rhabdomyosarcoma cells treated with atorvastatin (ATO) at 1/2 IC50 for 48 hours. (a-d) T-test analysis of differentially altered metabolites, with a chromatogram highlighting the desmosterol peak. (e) Lysine degradation pathway illustrating metabolite intervention by pre-incubation (24 hours) followed by atorvastatin treatment (½ IC50) for 72 hours. Dashed lines indicate multiple enzymatic steps in the pipecolate pathway; solid blue arrows represent the activity of the bifunctional enzyme AASS. (f-k) Viability assays of RD cells pre-incubated with specified metabolites for 24 hours, followed by atorvastatin treatment for 72 hours. Statistical significance was determined using two-tailed t-tests or one-way ANOVA with Dunnett’s post-hoc test. Significance levels: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.
Fig 3.
Metabolic supplementation alters western blot stress markers alone or combined with atorvastatin.
Representative Western blots for (a) mevalonate, (b) saccharopine, (c) aminoadipic acid, (d) lysine, (e) pipecolic acid, (f) α-ketoglutarate, (g) glutamic acid, and (h) lysine-p-nitroanilide (LPN) treatments (24 h), followed by atorvastatin treatment for 72 hours. All experiments were performed in duplicate from two independent experiments.
Fig 4.
LPN alters stress WB stress markers, OCR and ECAR parameters Western blot analysis of stress markers in RD cells treated with atorvastatin for 24 hours, with or without 0.01–0.1 mM LPN pre-treatment for 24 hours (a).
Densitometric analysis of BiP, FBX32, cleaved PARP, and Thr172-pAMPK levels in RD cells with or without 0.1 mM LPN pre-treatment for 24 hours. Densitometric analyses were conducted using ImageJ software, and bar graphs represent the mean ± SD of three independent replicates. Control levels were normalized to 1, and only statistically significant changes are shown (b-e). Mitochondrial respiration parameters in RD cells treated with 0.01–0.1 mM LPN, with or without atorvastatin at its IC50, for 24 hours (f). Glycolysis parameters in RD cells under the same conditions (g). Oligomycin-O, carbonyl cyanide-p-trifluoromethoxy phenyl-hydrazone-FCCP, rotenone/antimycin-R/A and 2-deoxyglucose- 2-DG. Statistical differences were assessed by one-way ANOVA followed by Tukey multiple comparison post hoc test. Significance levels compared to control: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05. Significance levels compared to atorvastatin treatment: ####p < 0.0001, ###p < 0.001, ##p < 0.01, #p < 0.05.
Fig 5.
LPN is metabolized to PNA and alters WB stress markers.
T-test analysis of (a) aminoadipic acid and (b) myo-inositol levels in RD cells treated with 0.01 mM LPN for 48 hours. Stability analysis of LPN based on absorbance at 405 nm in RD cells after 72 hours of incubation (c). Viability assays using crystal violet staining in RD cells treated with LPN or PNA for 72 hours (d). Viability assays of RD cells pre-incubated with PNA for 24 hours followed by atorvastatin treatment (1/2 IC50) for 72 hours (e). Western blot analysis of RD cells treated with LPN or PNA [0.01mM] for 24 hours followed by atorvastatin treatment for 48 hours (f). Statistical differences between treatments and controls were assessed using one-way ANOVA followed by Dunnett’s post-hoc test. Significance levels: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.