**3.3. Statins effects on respiratory chain complexes**

**3.1. Mitochondrial dysfunction caused by statins treatment**

392 Mitochondrial Diseases

drial antioxidant systems [132]. As a consequence, H<sup>2</sup>

our group in PC3 cells after simvastatin treatment [21, 22].

**3.2. Ca2+ and statins toxicity**

statins induced a direct oxidative damage in mitochondrial proteins [109].

Mitochondrial redox imbalance is associated with aging, degenerative disorders, and druginduced toxicity [26, 132]. Several reports concerning statin *in vitro* effects on isolated tissues or mitochondria from experimental models demonstrated that statins promote inhibition of mitochondrial respiration, mitochondrial oxidative stress, and cell death [47, 49, 109, 133]. It has been previously shown that lipophilic (cerivastatin, fluvastatin, atorvastatin, and simvastatin) and hydrophilic (pravastatin) statins-induced mitochondrial membrane potential decrease in rat skeletal muscle cell line (L6) [133]. The four lipophilic statins also induced mitochondrial swelling, cytochrome c release, and DNA fragmentation in these L6 cells. Mitochondrial β-oxidation enzymes activities were strongly impaired by all lipophilic statins, but in the case of pravastatin, it occurred only at high concentrations. In isolated rat skeletal muscle mitochondria, glutamatesupported state 3 respiration and respiratory control ratios were decreased by all lipophilic statins, but not by pravastatin [133]. According to the authors, this mitochondrial dysfunction caused by lipophilic statins in skeletal muscle might partially explain the muscle symptoms presented by some patients. Abdoli and coworkers demonstrated in isolated rat liver mitochondria that atorvastatin, simvastatin, and lovastatin increased ROS formation followed by lipid peroxidation, inner mitochondrial membrane depolarization, and a decreased GSH/GSSG ratio [47]. More recently, mitochondrial redox imbalance [67, 134] was observed in a genetic human familial hypercholesterolemia mouse model, the LDL receptor knockout mouse (*LDLr−/−)* [135]. Mitochondria isolated from several tissues of these mice (liver, heart, and brain) and intact spleen mononuclear cells presented higher ROS production and higher susceptibility to MPT. In addition, these mitochondria showed lower capacity to sustain reduced NADPH [67, 134], which is the most important reducing power involved in reconstituting mitochon-

O2

134]. Since cholesterol synthesis consumes a large amount of NADPH, we have proposed that the increased steroidogenesis observed in these mice would be partially responsible for the lower mitochondrial content of NADPH and Krebs cycle intermediates observed in their liver mitochondria [67, 134]. Therefore, we hypothesized that inhibition of cholesterol synthesis by statins treatment could prevent the decrease in NADPH oxidation in *LDLr−/−* mice mitochondria. Unexpectedly, liver mitochondria from wild type and *LDLr−/−* mice treated with lovastatin presented a higher susceptibility to PTP opening, and *in vitro* experiments revealed a drug dose- and class-dependence of this effect [109]. Statin induced PTP opening was shown to be Ca2+-dependent and associated with oxidation of protein thiol groups. Thus,

It has been proposed by our group and others that statins impair cellular Ca2+ homeostasis, leading to mitochondrial dysfunction. Increased cytosolic Ca2+ levels were observed after simvastatin treatment of myoblasts culture [136], rat skeletal muscle [137], and human skeletal muscle fibers, and this was followed by mitochondrial Ca2+ accumulation [138]. Indeed, Hattori and coworkers [139] proposed that statins induced Ca2+ release from the endoplasmic reticulum to the cytosol in human CD19+ primary lymphocytes. As a consequence of high Ca2+ levels in the cytosol, Ca2+ enters the mitochondria and induces MPT as demonstrated by

accumulates and PTP opens [67,

It is well known that enzymes containing 4Fe-4S clusters are particularly vulnerable to damage by superoxide or peroxynitrite radicals [140–145]. Complexes I and II present six and one of these 4Fe-4S clusters, respectively, thus showing a high superoxide-sensitivity. Some studies have demonstrated that superoxide generation inhibits respiration at complex I and II levels as a result of 4Fe-4S clusters damage. These alterations diminish resistance to Ca2+-induced MPT and induce necrotic cell death [65, 145]. As mentioned before, our group demonstrated that mitochondrial dysfunction caused by simvastatin incubation in permeabilized skeletal muscle was L-carnitine and CoQ10 sensitive [49]. L-carnitine did not protect against CoQ10 depletion, indicating that both CoQ10 and L-carnitine are protecting mitochondrial respiration due to its ROS scavenging properties. Since L-carnitine also binds Fe2+ [146], it is feasible that this antioxidant molecule interacts with 4Fe-4S clusters in complexes I and II of the respiratory chain, protecting these sites against superoxide attack. Simvastatin lowered the ADP-stimulated respiration supported by substrates of complexes I and II in primary human skeletal myotubes and increased susceptibility to MPT, mitochondrial oxidative stress, and apoptosis [48]. These results are in agreement with a decrease in complex I activity in muscle of patients undergoing statin treatment [147].

Another study performed in myoblasts culture (C2C12) incubated with several statins (atorvastatin, cerivastatin, fluvastatin, lovastatin, pitavastatin, pravastatin, rosuvastatin, and simvastatin) showed that the respiratory capacity is reduced not only at the levels of respiratory chain complexes I and II but also in complex III [148]. In this case, it was suggested that statins in the lactone form binds to Q<sup>o</sup> site of complex III, inhibiting its activity. Similarly, complex III activity of muscle from patients presenting myopathies induced by statins was also reduced [148]. On the other hand, statins do not seem to affect the complex IV-supported respiration [49, 148].
