**4. Cellular contractility in nonmuscle cells: the role of Rho and pleiotropic effects of statins**

In order to divide, migrate, and undergo tissue morphogenesis, cells change shape and exert forces either on the substrate that they are attached to or on the neighboring cells. Nonmuscle cells generate contractile stresses via molecular motors, such as myosin, that are able to convert chemical energy into mechanical work [2]. Myosin activity is controlled through phosphorylation of its light chain via myosin light chain kinase (MLCK) [100, 101] which, in turn, is activated by Rho kinase ROCK and the small GTPase Rho A upstream. In 1985, the Rho gene was first isolated, from abdominal ganglia of the Aplysia, and identified as a member of the Ras family [102]. In 1990, after injecting a constitutively active form of Rho (Vall4rho), Paterson and collaborators verified that active Rho is able to cause changes in cellular morphology inducing formation of stress fibers [103]. In 1992, Ridley and Hall showed that active RhoA induces formation of stress fibers and focal adhesions upon growth factor stimulation [104]. Being a GTPase, Rho can switch back and forth between its active state, when bound to GTP, and its inactive state, when bound to GDP. The switching process is finely regulated by guanine nucleotide exchange factors (GEFs), which promote activation, and GTPase-activating proteins (GAPs), which promote inactivation. There are approximately 60 GEFs and 70 GAPs that were already identified in the human genome [105]. In order to be activated, Rho goes through some posttranslational modifications that are essential to induce Rho migration toward the plasma membrane, where it gets activated. Prenylation is a posttranslational modification that is known to be pivotal for Rho translocation to the membrane [106]. Protein prenylation is essentially an insertion of a prenyl group, which is a hydrophobic group, to the c-terminal of a protein. That way, the protein has a lipid anchor that allows it to stay membrane bound. There are two types of prenylation: farnesylation and geranylgeranylation, which are regulated by farnesyltransferase and geranylgeranyltransferase I, respectively [107]. In the case of RhoA, the protein gets geranyl-geranylated before it goes to the membrane to get activated and trigger downstream effectors [108, 109] (**Figure 2**).

Both isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are synthetized by the mevalonate pathway. Interestingly, cholesterol is the end product of this pathway [110] (**Figure 3**). Therefore, by manipulating the mevalonate pathway, one can perturb both cholesterol synthesis and prenylation of important target proteins such as RhoA, and, as a consequence, cellular contractility (**Figure 3**).

The rate-limiting step of the mevalonate pathway is regulated by the enzyme HMG-CoA reductase. The activity of HMG-CoA reductase is precisely governed by the amount of cholesterol available. There are basically two different sources of cholesterol in the body: the exogenous one (obtained through intestinal absorption of cholesterol from the diet) and the endogenous one (through the *de novo* synthesis via the mevalonate pathway), being the endogenous source down regulated when enough cholesterol is obtained from nutrition [111]. During the early 1970s, a lot of effort was put into identifying pharmacological candidates that were able to reduce the HMG-CoA reductase activity especially in patients with high LDL cholesterol. In 1984, on a National Institutes of Health (NIH) Consensus Conference for Coronary Primary Prevention Trial, it was demonstrated the importance of a balance diet and drug treatment in order to lower LDL-cholesterol to prevent coronary heart disease [112]. After 1987, statins, that are essentially very specific drug inhibitors of HMG-CoA reductase activity, started to be prescribed for patients with high cholesterol

values [113]. By reducing the activity of HMG-CoA reductase, statins not only lower the amount of circulating cholesterol but also the amount of isoprenoid, both synthesized the mevalonate pathway, as one can see in **Figure 3**. Active RhoA as well as RhoA downstream effectors, such as ROCK, are inhibited upon statin treatment [114]. In endothelial cells, for example, a combination of flow and simvastatin exposure led to cell rounding and disorganization of the actin cytoskeleton [115]. In order to mimic atherosclerosis and aging effects on vessel walls, endothelial cells were plated in a series of substrates with low (physiological) and high stiffness values. High stiffness substrates increased both RhoA and ROCK activities. However, upon simvastatin incubation, contractility was abrogated in those cells

**Figure 3.** Simple schematic of the mevalonate pathway showing that posttranslational modifications pivotal for RhoA

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(red) activation and cholesterol (red) synthesis are part of the same intracellular pathway.

[116].

**Figure 2.** RhoA requires geranyl-geranylation in order to go to the membrane and be activated by RhoGEFs. Active RhoA triggers actomyosin contractility by inducing ROCK phosphorylation of myosin light chain (pMLC).

Role of Membrane Cholesterol in Modulating Actin Architecture and Cellular Contractility http://dx.doi.org/10.5772/intechopen.76532 43

it to stay membrane bound. There are two types of prenylation: farnesylation and geranylgeranylation, which are regulated by farnesyltransferase and geranylgeranyltransferase I, respectively [107]. In the case of RhoA, the protein gets geranyl-geranylated before it goes to

Both isoprenoids, farnesyl pyrophosphate and geranylgeranyl pyrophosphate, are synthetized by the mevalonate pathway. Interestingly, cholesterol is the end product of this pathway [110] (**Figure 3**). Therefore, by manipulating the mevalonate pathway, one can perturb both cholesterol synthesis and prenylation of important target proteins such as RhoA, and, as a

The rate-limiting step of the mevalonate pathway is regulated by the enzyme HMG-CoA reductase. The activity of HMG-CoA reductase is precisely governed by the amount of cholesterol available. There are basically two different sources of cholesterol in the body: the exogenous one (obtained through intestinal absorption of cholesterol from the diet) and the endogenous one (through the *de novo* synthesis via the mevalonate pathway), being the endogenous source down regulated when enough cholesterol is obtained from nutrition [111]. During the early 1970s, a lot of effort was put into identifying pharmacological candidates that were able to reduce the HMG-CoA reductase activity especially in patients with high LDL cholesterol. In 1984, on a National Institutes of Health (NIH) Consensus Conference for Coronary Primary Prevention Trial, it was demonstrated the importance of a balance diet and drug treatment in order to lower LDL-cholesterol to prevent coronary heart disease [112]. After 1987, statins, that are essentially very specific drug inhibitors of HMG-CoA reductase activity, started to be prescribed for patients with high cholesterol

**Figure 2.** RhoA requires geranyl-geranylation in order to go to the membrane and be activated by RhoGEFs. Active

RhoA triggers actomyosin contractility by inducing ROCK phosphorylation of myosin light chain (pMLC).

the membrane to get activated and trigger downstream effectors [108, 109] (**Figure 2**).

consequence, cellular contractility (**Figure 3**).

42 Cholesterol - Good, Bad and the Heart

**Figure 3.** Simple schematic of the mevalonate pathway showing that posttranslational modifications pivotal for RhoA (red) activation and cholesterol (red) synthesis are part of the same intracellular pathway.

values [113]. By reducing the activity of HMG-CoA reductase, statins not only lower the amount of circulating cholesterol but also the amount of isoprenoid, both synthesized the mevalonate pathway, as one can see in **Figure 3**. Active RhoA as well as RhoA downstream effectors, such as ROCK, are inhibited upon statin treatment [114]. In endothelial cells, for example, a combination of flow and simvastatin exposure led to cell rounding and disorganization of the actin cytoskeleton [115]. In order to mimic atherosclerosis and aging effects on vessel walls, endothelial cells were plated in a series of substrates with low (physiological) and high stiffness values. High stiffness substrates increased both RhoA and ROCK activities. However, upon simvastatin incubation, contractility was abrogated in those cells [116].

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]

as well as adhesion energy between membrane and cytoskeleton, which decreased the lipid diffusion coefficient [119]. Serum starvation followed by cyclodextrin-mediated cholesterol depletion increased stress fiber formation and RhoA activation in an osteoblast cell line [120]. Later on, those results were also corroborated by our group in a murine fibroblast cell line [41]. Similar features can also be observed in an osteosarcoma cell line U2OS (**Figure 4**) in which cholesterol depletion led to stress fiber formation and reorganization of actin cytoskeleton. More studies need to be performed in order to understand why different manipulations in

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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

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

cholesterol content trigger opposite results regarding cellular contractility.

cytoskeleton disruption and apoptosis of smooth muscle cells [123].

**sarcomeres and cholesterol**

contractility in several levels.

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

**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 the latter. Scale bar 10 μm.

as well as adhesion energy between membrane and cytoskeleton, which decreased the lipid diffusion coefficient [119]. Serum starvation followed by cyclodextrin-mediated cholesterol depletion increased stress fiber formation and RhoA activation in an osteoblast cell line [120]. Later on, those results were also corroborated by our group in a murine fibroblast cell line [41]. Similar features can also be observed in an osteosarcoma cell line U2OS (**Figure 4**) in which cholesterol depletion led to stress fiber formation and reorganization of actin cytoskeleton. More studies need to be performed in order to understand why different manipulations in cholesterol content trigger opposite results regarding cellular contractility.
