**5. Cellular contractility in muscle cells: interplay among Ca2+, sarcomeres and cholesterol**

Even though statins had been shown to be relatively safe and to promote health benefits to patients with high risks of cardiac diseases, there are some side effects and risks associated with statin therapy. Myotoxicity is one of the most adverse side effects, being the most common clinical outcomes: myosite, myalgia, and rhabdomyolysis [121]. In vitro studies performed on single muscle fibers isolated from rat skeletal muscle showed that fluvastatin and pravastatin led to contractility impairment and vacuolization of the muscle after 72 h of treatment and cell death after 120 h. Those changes in cellular morphology and contraction were proven to be dependent on geranyl-geranylation of GTPases since concomitant incubation of fluvastatin and geranylgeranyl pyrophosphate attenuated the deleterious effects of statins [122]. In vivo and in vitro treatment with simvastatin also led to contractile dysfunction, actin cytoskeleton disruption and apoptosis of smooth muscle cells [123].

Regarding the effects of direct cholesterol depletion mediated by MβCD on muscle cells, our group demonstrated, using primary cell culture of neonatal rat cardiomyocytes, that a lower cholesterol content increased the contraction rate of those cells and also led to defects in cell relaxation [8]. Moreover, cholesterol depletion increased the Ca2+ cytoplasmic concentration and Ca2+ sparks during contraction. This phenotype can be attributed to changes in caveolin3 and L-type Ca2+ channels distribution across the plasmalemma and hyperactivation of cAMP-dependent PKA activity. Cholesterol-depleted cardiomyocytes also present aberrant myofibrils due to calpain (a Ca2+ sensitive protease) activation. By using high-quality confocal microscopy and quantitative data analysis, this work has set in stone the role of cholesterol in regulating cardiomyocyte contractile behavior [8]. Other groups have also shown, for adult rat cardiomyocytes, that cholesterol depletion due to MβCD incubation changed localization of caveolin-3 from a raft to a nonraft membrane fraction changing MAPK signaling and increasing contractility and intracellular Ca2+ concentration [124]. Adult murine cardiomyocytes treated with MβCD also presented impairment in the T-tubule system and intercalated discs, which reinforces the role of cholesterol in regulating cardiac contractility [125]. More studies need to be performed in order to understand why MβCD-driven cholesterol depletion in nonmuscle cells increase contractile behavior whereas in muscle cells the same treatment tend to abrogate cellular contractility in several levels.

**Figure 4.** Fixed human osteosarcoma cell line U2OS, labeled for actin (red) paxillin (cyan), a focal adhesion protein. Notice the change in stress fibers between control and cholesterol depleted cells and how aligned the stress fibers get in

Interestingly, when cholesterol is directly depleted by MβCD, an opposite trend is observed regarding nonmuscle cellular contractility. Human skin fibroblasts, after MβCD treatment, showed a reduction in the mobility of plasma membrane proteins being that reduction in motion a direct result of cytoskeleton reorganization [117]. It was also shown, for bovine aortic endothelial cells, that MβCD-dependent cholesterol depletion increased cortical stiffness [118]

the latter. Scale bar 10 μm.

44 Cholesterol - Good, Bad and the Heart
