Statin-induced toxicity in human skeletal muscle cells

Inhibition of the HMG-CoA-reductase results in reduced endogenous cholesterol synthesis which in turn leads to an up-regulation of LDL receptors and thereby ultimately lowers the circulating level of cholesterol. This therapeutic concept is successfully used in patients with hyperlipidemia. While HMG-CoA-reductase inhibitors (statins) are well tolerated, some patients suffer from myoalgia and myositis. Especially, the combination of HMG- CoA-reductase inhibitors with e.g. fibrates (gemfibrozil) in humans increases the risk of myositis and rhabdomyolysis, a potentially life-threatening complication. The molecular mechanisms by which HMG-CoA- reductase inhibitors cause apoptosis and/or necrosis exclusively in skeletal muscle, are not well understood. The goal of the current project is to elucidate the mechanism by which statins damage skeletal muscle cells. Exposure of cultured primary human skeletal muscle cells (undifferentiated satellite cells or differentiated myotubes) to HMG-CoA-reductase inhibitors will be used to activate the induction of apoptosis. HMG-CoA- reductase inhibitors are capable to induce Ca2+ transients immediately after application to human skeletal muscle cells. The project intends to verify the working hypothesis that elevations of myoplasmic Ca2+ concentrations induced by statins and apoptosis of skeletal muscle cells are causally related. Therefore, we shall delineate the signalling cascade(s) that are affected by statins. We shall define: (i) the nature of the Ca2+ pools controlled by statins and its relation to apoptosis (ii) the contribution of cholesterol depletion and altered isoprenylation of signalling proteins (iii) the trigger for the apoptotic signalling cascade by employing a combination of cell biological approaches and techniques of pharmacology and biochemistry. It is expected that this work will generate new insights into the pathophysiology underlying rhabdomyolysis. These ought to ultimately make it possible to improve the safety of statins and devise new therapeutic approaches to prevent and treat muscle diseases.

The 3-Hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) are widely used and well tolerated cholesterol-lowering drugs. In rare cases side effects occur in skeletal muscle, including myositis or even rhabdomyolysis. However, the molecular mechanisms are not well understood that lead to muscle-specific side effects. Here we show that statins cause apoptosis in differentiated human skeletal muscle cells. The prototypical representative of statins, simvastatin, triggered sustained intracellular Ca2+ transients leading to calpain activation. Intracellular chelation of Ca2+ completely abrogated cell death. Moreover, ryanodine also completely prevented the simvastatin induced calpain activation (Sacher et al., 2005). Downstream of the calpain activation simvastatin led to a translocation of Bax to mitochondria in a caspase 8 independent manner. Consecutive activation of caspase 9 and 3 execute apoptotic cell death. These data delineate the signalling cascade that leads to muscle injury caused by statins and helps to explain why physical exercise aggravates muscle injury. The fact that the ryanodine receptor inhibitor, ryanodine, prevents calpain activation prompted us to screen for novel ryanodine receptor antagonists. With the suramin analogue, NF676 we characterised a novel lead compound of use-dependent ryanodine receptor antagonists (Wolner et al., 2005). This compound is currently tested in the cardiac system to evaluate its potential therapeutic effect in arrhythmia and end stage heart failure. We have also expanded our knowledge on the statin-induced apoptosis in tumor cells like human rhabdomyosarcoma RD cells. Here we show that statins activate the mitochondrial pathway of apoptosis via translocation of Bax from the cytosol to mitochondria (Werner et al., 2004). The prototypical representative of statins, simvastatin, induced consecutive activation of caspase 9 and 3 in a concentration dependent manner. Interestingly, statins did not trigger a Ca2+ transient or calpain in these cells. Simvastatin treated RD cells could be completely rescued from apoptosis by the co-application of mevalonic acid, indicating that deprivation of cholesterol precursors is essential for statin induced apoptosis. However, pre-treatment with subthreshold concentrations of simvastatin were sufficient to augment doxorubicin toxicity via the mitochondrial apoptotic machinery (Werner et al., 2004). Although the molecular mechanisms of such a mutual amplification in drug action are not clear at the moment, it is of potential interest for future co-administration of statins in clinical cancer treatment.