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

Abstract

The present study investigated the cytotoxic effects of statins (atorvastatin (ATR) and simvastatin (SIM), resp.) and methyl-beta-cyclodextrin (MβCD), at their respective IC50 concentrations, on muscle regeneration in the in vitro model of murine C2C12 myoblasts. Cotreatment with mevalonate (MEV), farnesol (FOH), geranylgeraniol (GGOH), or water-soluble cholesterol (Chol-PEG) was employed to determine whether the statin-dependent myotoxicity resulted from the lower cholesterol levels or the attenuated synthesis of intermediates of mevalonate pathway. Our findings demonstrated that while GGOH fully reverted the statin-mediated cell viability in proliferating myoblasts, Chol-PEG exclusively rescued MβCD-induced toxicity in myocytes. Statins caused loss of prenylated RAP1, whereas the GGOH-dependent positive effect was accompanied by loss of nonprenylated RAP1. Geranylgeranyltransferases are essential for muscle cell survival as inhibition with GGTI-286 could not be reversed by GGOH cotreatment. The increase in cell viability correlated with elevated AKT 1(S463) and GSK-3β(S9) phosphorylations. Slight increase in the levels of autophagy markers (Beclin 1, MAP LC-3IIb) was found in response to GGOH cotreatment. Autophagy rose time-dependently during myogenesis and was inhibited by statins and MβCD. Statins and MβCD also suppressed myogenesis and neither nonsterol isoprenoids nor Chol-PEG could reverse this effect. These results point to GGOH as the principal target of statin-dependent myotoxicity, whereas plasma membrane cholesterol deposit is ultimately essential to restore viability of MβCD-treated myocytes. Overall, this study unveils for the first time a link found between the GGOH- and Chol-PEG-dependent reversal of statin- or MβCD-mediated myotoxicity and cytoprotective autophagy, respectively.

Geranylgeraniol prevents statin-induced skeletal muscle fatigue without causing adverse effects in cardiac or vascular smooth muscle performance

“…This investigation has verified findings from in vitro studies 22,27,30,32 and demonstrated that supplementation with GGOH can prevent statin-induced skeletal muscle fatigue in rodents. Gastrocnemius muscles isolated from the SIM group exhibited a significant decline in muscle force production, an effect which is characteristic of this rodent model as well as statininduced myalgia in clinical practice.…”

“…GGPP was administered as its precursor, GGOH, owing to its greater cell/tissue permeability. 32 GGOH was dissolved in saline and delivered at a dose of 15 mg kg À1 day À1 . This concentration was used as it was calculated to be an equivalent mid-range dose (per kg bodyweight) to that which has previously been delivered to rats.…”

“…[21][22][23][24][25][26][27][28][29] Several in vitro studies have demonstrated an association between GGPP depletion and statin-induced damage in skeletal myofibers/myocytes. 21,22,[30][31][32] Moreover, these investigations have also shown that supplementation with GGPP (or its precursor, geranylgeraniol [GGOH]), can prevent this myotoxicity.…”

The Many Faces of Rap1 GTPase

“…Impaired prenylation of the Rap1 protein leads to lower muscle cell viability (repressed protein synthesis) and impeded autophagy. This mechanism might explain muscle damage and hampered myogenesis in statin-dependent myopathies [ 65 ].…”

“…Consequently, the regeneration and growth of skeletal muscle is inhibited. Our studies on C2C12 cells show that the Rap1, which becomes active due to prenylation with geranylgeraniol, plays a key role in the IGF-1/PI-3K/Akt1 signaling pathway [ 65 ].…”