**8. Ca2+ homeostasis and statin-induced myopathy**

Acute application of simvastatin on human skeletal muscle fibers led to a large release of Ca2+ into the sarcoplasm [76]. The authors showed that mitochondrial Ca2+ efflux through both permeability transition pores (PTP) and Na+ /Ca2+-exchanger (NCE) was a major initiator of the large sarcoplasmic reticulum (SR) Ca2+ release by affecting ryanodine receptor 1 (RyR1, Figure 11). Furthermore, the effects of simvastatin on Ca2+ homeostasis were not linked to the disturbed cholesterol synthesis pathway as GGPP and FPP treatments did not prevent statininduced Ca2+ waves. They were caused by simvastatin-dependent fall in mitochondrial membrane depolarization and Ca2+ efflux to the cytoplasm. Next, Ca2+ was recaptured by the SR that in turn triggered the SR release of Ca2+ by at present unknown molecular mechanism as "calcium-induced Ca2+ release" is implausible since the main RyR isoform in mammalian skeletal muscle is poorly sensitive to Ca2+ [77]. It is apparent from this study that mitochondria played critical role, and that Ca2+ efflux from mitochondria resulted from alteration of mitochondrial respiratory chain, as mitochondrial membrane potential (MMP) decreased with concomitant rise in NADH concentration. Nonetheless, with regard to these experiments some criticism has to be reserved, as simvastatin concentrations (50–200 µM) were much above the pharmacological range. Summing up, these observations point to ubiquitin/proteasome (UP) proteolysis and mitochondria as skeletal muscle target of statins, while the exact nature of their detrimental action (direct or indirect) remains to be elucidated. Recently, some interesting data were obtained from transcriptomic analysis of biopsies collected from atorvastatin-treated and exercised vs. nonexercised skeletal muscles of healthy volunteers. The authors complement severalfold rise in UP pathway gene expression in 8-hours eccentrically exercised vastus lateralis muscles baseline compared to the right leg after statin/placebo treatment [78].

**Figure 11.** A possible direct pathophysiological effect of statins. Although statin myotoxycity may occur through the reduction of cholesterol synthesis, a direct effect of statins has been reported in vitro and in vivo in muscle fibers from both animal and human models. This diagram summarizes some of the most recent data suggesting a pathophysiolog‐ ical mechanism. Statins diffuse into muscle fibers and inhibit complex I of the mitochondrial respiratory chain (RC). This depolarizes the inner membrane (Dc) triggering a calcium release through the permeability transient pore (PTP) and sodium-calcium exchanger (NCE). This results in a first elevation of cytoplasmic calcium that will be partially up‐ taken by the sarcoendoplasmic reticulum calcium pump (SERCA) to the sarcoplasmic reticulum (SR). When overload‐ ed, SR may spontaneously release calcium through the ryanodine receptor (RyR1) to generate a calcium wave. A direct effect of statins on RyR1 may not be excluded (dotted line). Impaired mitochondrial function and consequently calci‐ um signaling may account for muscle symptoms, reprinted from Sirvent et al. 2008 [48].

Deregulation of calcium ion (Ca2+) homeostasis in mitochondria is indicated as initial step in cascade of events leading to statin myopathy [76, 79]. In several experiments carried out on C2C12 myoblasts and human muscle biopsies, statins impaired mitochondrial respiration and sensitized muscle fibers to calcium signaling. Accordingly, muscle fibers showed reduced level of ATP and higher frequency of Ca2+ waves [46, 79]. Disturbed regulation of Ca2+ homeostasis (elevated cytoplasmic concentrations) is known to trigger activity of calpains [80], whereas raise in ROS is associated with caspase cascade [81–82]. As RSC are enriched in SM, they obviously should have high LR/C representation. Modulation of SL modifications with sphingomyelinase inhibitors would provide closer look on LR/C impact cell viability/morbid‐ ity. Next to statins widely used as hypocholesteremic drugs, polyunsaturated fatty acids (PUFA) are frequently recommended to lower blood plasma concentration of low density lipoproteins (LDL) and triglycerides (TG). There is evidence that PUFA (n–3) synergize with statins in their positive effect, furthermore some PUFA (EPA and DHA) prevent statin-induced myopathy [83–84]. It was found that statins evoke endoplasmic reticulum stress (ERS) and ERS inhibitors as well as PUFA attenuate this response most likely through PPARγ-dependent mechanism. Detailed outline of PUFA protective effect has, however, not been explained. Scientific problem is to find out if LR contribute to the activation of RSC. Furthermore, if LR/C are essential to recruit RSC to enter the differentiation program, what would have been if LR are ablated? The study aimed to shed more light on the side effects of statin administration to skeletal muscle is urgently needed. Novelty of research should address etiology to abnormal function of sarcolemma in skeletal muscle cells of statin-treated subjects. We found only one report showing that CHOL depletion impaired muscle function [17]. CHOL is essential in holding together lateral assemblies of lipids in LR nanodomains. The latter are indispensable to cell signaling as they form platforms for signalosomes (proteins assembled in order to provide signal transduction from PM receptors). Their role has been corroborated for PI3- K/AKT and JAK/STAT but not Ras/Raf/MEK/ERK signaling pathway. We assume that former cascades (PI3-K/AKT and/or JAK/STAT) are involved in RSC activation while the latter are important to initiate proliferation. As muscle cell differentiation proceeds, the representation of SL in LR is subjected to additional modifications [85–88]. We guess these changes are related to the activation of sphingomyelinases and other SL converting enzymes. Ceramides and sphingosines were reported to frequently affect cell functions including proliferation, differ‐ entiation, and viability [89, 90], and other bioactive lipids are also important players in muscle growth and regeneration. At the same time, involvement of ROS and mitochondria, Ca2+ homeostasis, and proteolytic systems should be examined.
