Geranylgeraniol Prevents Statin-Dependent Myotoxicity in C2C12 Muscle Cells through RAP1 GTPase Prenylation and Cytoprotective Autophagy

Figure 5

Effect of nonsterol isoprenoids and soluble cholesterol treatments on acidic vacuolar organelles (AVO) as red to green fluorescence intensity ratio (R/GFIR) in C2C12 myoblasts affected by statins or MβCD. C2C12 myoblasts were exposed for 24, 72, or 120 h to statins or MβCD (IC₅₀) (day 1—proliferating myoblasts, day 3—differentiating myotubes, and day 5—differentiated myotubes). ATR or SIM was administered in the listed concentration at each day of differentiation: ATR: day 1—76 μM, day 3—46 μM, and day 5—36 μM; SIM: day 1—87 μM, day 3—6 μM, and day 5–3 μM. MβCD was added to give the final concentration: day 1—2.7 mM, day 3—1.9 mM, and day 5—1.1 mM or vehicle control (0.1% DMSO or 2% HS DMEM) without or with selected mevalonate pathway intermediate (mevalonate 100 μM, geranylgeraniol 10 μM, farnesol 10 μM, Chol-PEG 1 mM, dolichol 1 μg/mL, and ubiquinol 10 μg/mL). Next, cells were subjected to vital staining with acridine orange (see Materials and Methods). Red to green fluorescence intensity ratio (R/GFIR) was calculated in at least 10 replicates for each treatment and nontreated controls. Two-way ANOVA test for R/GFIR followed by Bonferroni’s multiple comparisons was employed to analyze the data. The results of [time (proliferating myoblasts, differentiating myotubes, differentiated myotubes)] amounted to , for ATR; , for SIM; and , for MβCD. Treatment: ATR, ATR + MEV, ATR + GGOH, ATR + FOH, and ATR + Chol-PEG (, ); SIM, SIM + MEV, SIM + GGOH, SIM + FOH, and SIM + Chol-PEG (, ); MβCD, MβCD + MEV, MβCD + GGOH, MβCD + FOH, and MβCD + Chol-PEG (, ). Interaction: , for ATR; , for SIM; , for MβCD. Error bars = SEM and , , and for comparison between the means. Results are means of three independent experiments.

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