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.
Highlights
Atherosclerosis and cardiovascular diseases are main causes of mortality in humans [1]
The effect of the different experimental factors tested (mevalonate (MEV), geranylgeraniol (GGOH), farnesol (FOH), dolichol (DOH), ubiquinol (UBOH), and cholesterol as watersoluble conjugated Chol-poly(ethylene-glycol 600) (PEG)) in cell cultures challenged with statins or MβCD at their half-maximal cell viability inhibitory concentration (Supplementary data 1) was monitored via the MTT assay at days 1, 3, and 5 of myogenesis
We examined the differential effect of statins (ATR, SIM) and the cholesterol chelator (MβCD) in skeletal muscle cells undergoing differentiation process divided into three basic steps: postmitotic proliferation, cell fusion, and myotube enlargement
Summary
Atherosclerosis and cardiovascular diseases are main causes of mortality in humans [1]. Despite exhibiting various pleiotropic beneficial effects including anti-inflammatory, antithrombogenic, antisclerotic, antiosteoporotic, and anticancer properties [4], some varieties of statins induce skeletal muscle injury including myalgia, myositis, and lifethreatening rhabdomyolysis [5]. The mechanism(s) leading to statin myotoxicity is unknown existing data indicate the association of the drugs with vacuolation of skeletal muscle fibers, blebbing of sarcolemma and cell necrosis [6]. Geranylgeraniol (GGOH) seems a good candidate as few reports showed the beneficial role of the compound in rescuing statin-induced myopathy [6, 7] work by other authors has questioned the causal relationship between muscle safety and protein isoprenylation [8]. In spite of the beneficial effects of GGOH, the molecular mechanisms behind its cytoprotective action remain to be elucidated
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