Cerivastatin induces type-I fiber-, not type-II fiber-, predominant ...

The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.36, No.4, 445-452, 2011

445

Original Article

Cerivastatin induces type-I fiber-, not type-II fiber-, predominant muscular toxicity in the young male F344 rats Hisakuni Obayashi1, Yoshikazu Nezu1, Hatsue Yokota1, Naoki Kiyosawa2, Kazuhiko Mori2, Naoyuki Maeda1, Yoshiro Tani1, Sunao Manabe3 and Atsushi Sanbuissho2 Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, Japan 2Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., 717 Horikoshi, Fukuroi, Shizuoka 437-0065, Japan 3Daiichi Sankyo Co., Ltd., 3-5-1 Nihonbashi Honcho, Chuo-ku, Tokyo 103-8426, Japan 1

(Received March 30, 2011; Accepted June 17, 2011)

ABSTRACT — 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins) are associated with adverse skeletal muscle toxicity, but the underlying mechanism remains unclear. To investigate the pathological mechanism of statin-induced myotoxicity, cerivastatin (20 ppm; corresponding to 2 mg/kg/day) was dietarily administered to young male F344 rats for 10 days, and time-course clinical observations, measurement of plasma creatine kinase activity, and light and electron microscopy of type I fiber-predominant skeletal muscle (soleus) or type II fiber-predominant skeletal muscles (extensor digitorum longus and tibialis anterior), were performed. Clinical symptoms including weakness of hind limbs, staggering gait and body weight loss, accompanied by marked plasma creatinine kinase elevation in rats fed cerivastatin at around Day 6 to 8. Interestingly, microscopic examination revealed that cerivastatin-induced muscle damages characterized by hypercontraction (opaque) and necrosis of the fibers were of particular abundance in the soleus muscle at Day 8, whereas these histological lesions in the extensor digitorum longus and tibialis anterior were negligible, even at Day 9. Prior to manifestation of muscle damage, swollen mitochondria and autophagic vacuoles in the soleus were observed as the earliest ultra structural changes at Day 6; then activated lysosomes, disarray of myofibril and dilated sarcoplasmic reticulum vesicles became ubiquitous at Day 8. These results demonstrate that cerivastatin induces type I fiber-predominant muscles injury, which is associated with mitochondrial damage, in young male F344 rats. Since the rat exhibiting type I fiber-targeted injury is a unique animal model for statin-induced myotoxicity, it will be useful for gaining insight into mechanisms of statin-induced myotoxicity. Key words: Cerivastatin, Rat, Myotoxicity, Soleus, Extensor digitorum longus, Mitochondria INTRODUCTION 3-Hydroxy-3-methylglutaryl coenzyme A reductase inhibitors, commonly referred to as statins, are generally regarded as safe and well-tolerated in patients. However, statins are also associated with skeletal muscle-related toxicity ranging from mild myopathy such as myositis and myalgia to severe rhabdomyolysis. The incidence rate of myotoxicity was relatively low and life-threatening rhabdomyolysis was rare for this class of drugs with monotheraphy (Thompson et al., 2003; Rosenson 2004); however cerivastatin (CER) was voluntarily withdrawn from the market because of unexpectedly high incidenc-

es of adverse effects with approximately 100 rhabdomyolysis-related deaths with monotherapy and sometimes in combination with gemfibrozil (Farmer 2001; Staffa et al., 2002). Although the exact mechanism of how statins induce myotoxicity has not been fully understood, a variety of statins were reported to induce myotoxicity in animal species in rats (Smith et al., 1991; Waclawik et al., 1993; Reijneveld et al., 1996; Schaefer et al., 2004; Westwood et al., 2005, 2008). In addition, the potential degree of statins’ myotoxic risk is reportedly related to be associated with their physicochemical properties such as lipophilicity and thus associated variable kinetic behavior including different tissue selectivity (Rosenson, 2004).

Correspondence: Hisakuni Obayashi (E-mail: [email protected])

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446 H. Obayashi et al.

Skeletal muscle fibers in mammalian can be classified into two major types (i.e., type I and type II), and are consisted of several isoforms of myosin heavy chain (MHC): two developmental isoforms, one slow isoform (type I fiber; MHC I/β) and three fast isoforms (type II fibers; MHC IIA, IIX/IID and IIB) (Pette and Staron, 2000). Several investigators have demonstrated that statin-induced muscle necrosis was primarily on type II fibers, with little effects on type I fibers (Smith et al., 1991; Waclawik et al., 1993; Reijneveld et al., 1996; Schaefer et al., 2004; Westwood et al., 2005, 2008). Westwood et al. (2005, 2008) reported that statininduced “muscle fiber-type selective toxicity” was suggested to match their cellular oxidative/glycolytic nature, with glycolytic type IIB fiber being the most vulnerable to statin-induced myotoxicity (Westwood et al., 2005, 2008). On the other hand, other factors such as age, gender and hormone have also been shown to influence characteristic of the composition and expression level of the isoforms with skeletal muscles (Degens et al., 1998; Biral et al., 1999; Pette and Staron, 2000), which may affect sensitivity of statin-induced myotoxicity. In fact, myopathic signatures due to statins were different among ages or genders as well as disparate skeletal muscles (Reijneveld et al., 1996; Schaefer et al., 2004; Westwood et al., 2005). Based on this information, it is necessary to understand the precise pathogenic mechanisms underlying the statin-elicited myotoxicity by further characterizing the statin-induced fiber type-specific myotoxicity with diverse experimental conditions. To accomplish this objective, we examined pathogenic mechanism of CER-induced myotoxicity using young rats, which are reported to be more susceptible to statin-induced muscular injury than adult rats (Reijneveld et al., 1996). We used male rats because they are more sensitive to muscle damage than females (Amelink and Bär, 1986). MATERIALS AND METHODS Test substance CER was synthesized by Chemtec Labo., Inc. (Tokyo, Japan). CER was mixed with powdered ingredients of a purified diet (AIN-93G, Oriental Yeast Co., Ltd., Tokyo, Japan) with concentration of 20 ppm, and the mixture was pelleted and gamma sterilized at 15 kGy. Animals and housing condition Male F344/DuCrj rats were purchased from Charles River Japan, Inc. (Kanagawa, Japan). Animals were housed in an air-conditioned rooms (temperature, 21.0 to 25.0°C; relative humidity, 45.0 to 65.0%; light cycle, Vol. 36 No. 4

12 hr (7:00 to 19:00)/day; ventilation, 12 to 15 changes filtered/hr) and given commercial laboratory chow (AIN-93G, Oriental Yeast Co., Ltd.) and tap water ad libitum. In all experiments, animals were subject to statin intake from the age of 4 weeks. All experimental protocols were approved by the Ethics Review Committee for Animal Experimentation of Sankyo Co., Ltd. (Tokyo, Japan). Study design The animals were divided into two experiments. In Experiment 1, time-course clinical observations, and measurements of body weight, food consumption and plasma creatine kinase (CK) activity were conducted to determine the optimal time point for histopathology examination. Experiment 2 was conducted to characterize histopathological signatutre of the CER-induced myotoxicity. Based on our preliminary dose-range finding study, the concentration of CER was set as 20 ppm (equivalent to 2 mg/kg/day dose level), which caused skeletal myopathy without any acute phase antemortem findings. CER intake was daily confirmed by measuring body weight and food consumption in both experiments. The examination items and its schedule in each experiment were as follows: Experiment 1: Rats consisting of 5 animals per group were fed with 20 ppm CER-mixed diet or commercial diet only as controls for 10 days. During the experiment, clinical observations, and measurement of body weight and food consumption were daily performed. Blood sampling for CK measurement was carried out on Days 3, 6, 7, 8, 9, and 10. Experiment 2: Rats consisting of 5 animals per group were fed with 20 ppm CER-mixed diet or commercial diet only as controls for 9 days. During the experiment, clinical observation, and measurement of body weight and food consumption were daily performed. Histopathologic examination were conducted on Days 6, 8, and 9. Measurement of plasma CK activity Blood samples were obtained from all animals via tail vein at each scheduled time point. Blood was heparinized and centrifuged to obtain plasma samples. Plasma CK activity was measured by a dry-chemistry method (Fuji DRI-CHEM 3500V, Fuji Film Medical Co., Ltd., Tokyo, Japan). Light microscopy The animals examined in Experiment 2, were euthanized under ether anesthesia at each scheduled time point, and the soleus, tibialis anterior and extensor digitorum longus

447 Cerivastatin-induced Type I fiber-predominant myotoxicity in rats

(EDL) muscles were excised and flash-frozen in isopentane chilled to -150°C. The frozen sections, 10 μm thick, were processed for staining with hematoxylin and eosin. Muscle histochemistry The frozen sections were stained with NADH-tetrazolium reductase (NADH-TR), ATPase (preincubation at pH 4.2, 4.6 and 10.7) and modified Gomori trichrome. To semi-quantitative evaluate the CER-induced myopathic findings, NADH-TR cross sections were scored according to the following definition: 0, no abnormalities; 1, large granules and disarray of myofibrils; 2, larger granules and barely identifiable myofibrils; and 3, coagulation of granules and unidentifiable myofibrils. Total scores for 1,000 muscle fibers were calculated and designated as “NADHscore”. Modified Gomori trichrome staining was performed to evaluate whether or not ragged-red fibers are observed in the case of mitochondrial myopathy (Riggs et al., 1986). Electron microscopy The soleus specimens were fixed with 2.5% glutaraldehyde buffered with 0.1 M cacodylate buffer (pH 7.2). The soleus were further postfixed in 2% osmium tetroxide, dehydrated in graded alcohols and embedded in epoxy resin. Semi-thin sections (1-2 μm) were stained with toluidine blue stain for the light microscopic examination. Ultrathin sections were stained with uranyl acetate and lead acetate, and were examined using a Hitachi H-7500 transmission electron microscope (Hitachi High-Technologies Corporation, Tokyo, Japan).

Fig. 1.

Statistical analysis Data on the body weight and plasma CK activity were expressed as the mean ± S.D. or range. These quantitative data were further analyzed by an F-test, followed by Student’s t-test or Aspin-Welch’s t-test between CER-treated and the concurrent control groups. The Spearman rankcorrelation coefficient (r) was used to define a relationship between plasma CK activity and NADH-score. A P value less than 0.05 was considered statistically significant. These statistical analyses were performed by the use of SAS® System Release 8.2 (SAS Institute Inc., Cary, NC, USA). Food consumption per animal (g/rat/day) was calculated by subtracting the remaining food weights from the food supplied for each group, and statistical analysis was not performed. RESULTS Clinical observation, body weight, food consumption, and plasma CK activity of CER treated rats (Experiment 1) Time-course observation of general condition was performed in the Experiment 1. CER treatment showed significant decrease in body weight from Day 6, and marked decrease in food consumption on Day 8 (Fig. 1). On Day 8, two of the five rats fed with CER developed serious staggering gait and trailing with hind limbs indicative of muscle weakness in the extremities. In addition, they showed hypoactivity and labored respiration, and were humanely sacrificed accordingly. The remaining three rats fed with CER showed slight muscle weakness in one or both hind limbs on Day 8, and showed serious mus-

Time-course changes in body weight, food consumption and plasma CK level (Experiment 1). (A) Body weight, (B) food consumption, (C) plasma CK level in control (○) and CER-treated rats (●). Body weight and plasma CK level, data represent the mean values ± S.D. of 1 to 5 rats (a, n = 3; b, n = 1 because of death). * P < 0.05, ** P < 0.01: compared with the control group by a Student’s t-test. # P < 0.05, ## P < 0.01: compared with the control group by an Aspin-Welch’s t-test. For data of food consumption, data per animal (g/rat/day) of 1 to 5 animals (a, n = 3; b, n = 1 because of death) was calculated by subtracting the remaining food weights from the food supplied for each group. Vol. 36 No. 4


cle weakness in the extremities on Day 9. Two of the three rats became moribund and were sacrificed on Day 9, but the one remaining rat survived to Day 10. Plasma CK activity was markedly elevated in CER-treated rats at around Day 8 and the CK elevation was generally associated with the occurrence of serious myopathic signs. Histopathology examination of CER-induced myotoxicity (Experiment 2) Light and electron microscopic examinations were performed in Experiment 2 to characterize detailed histopathological characteristics of the CER-induced myotoxicity. In Experiment 2, CER-treated rats also showed decreases in body weight and food consumption similar to those of Experiment 1 (data not shown). Microscopic examination revealed that there were no findings in all of the muscles in CER-treated rats necropsied on Day 6. On Day 8, hyper-contracted (opaque) and variable in size and fiber necrosis were evident in the soleus muscle of rats fed with CER (Fig. 2C), whereas tibialis anterior (Fig. 2D) and EDL muscles in the same rats were relatively preserved. Histochemical staining with NADH-TR demonstrated that a small number of dense NADH-positive granules and disarray of myofibrils were present in the soleus and EDL muscles of 2 and 1 respectively CERtreated rats, whereas tibialis anterior muscles were not affected on Day 6 (Table 1). On Day 8, dense NADHpositive granules and coarse-meshed intermyofibrillar

network were abundant in the soleus muscle of 3 rats given CER (No. 11, 14 and 15 in Table 1). Similar findings were observed in the tibialis anterior and EDL muscles in these rats, but it was of relatively minimal severity and infrequent (Fig. 3 and Table 1). In the remaining 2 rats fed CER, all of the muscles were almost intact (No. 12 and 13 in Table 1). The NADH staining pattern revealed that muscle damages in the soleus and tibialis anterior were closely correlated with CK elevations (soleus: r = 0.76, P < 0.01; tibialis anterior: r = 0.74, P < 0.01,), whereas that of EDL did not show apparent correlation (r = 0.45, statistically not significant). Gomori trichrome and ATPase staining did not indicate any CER-related changes in all of the muscles (data not shown). Electron microscopic examination revealed that a number of swollen mitochondria, activated lysosomes, inclusion body in mitochondria and autophagic vacuoles were present in the soleus muscle of the rat treated with CER sacrificed on Day 6 (Fig. 4B). In addition, electrondense mitochondria, autophagic vacuoles ingesting membranous organelles between the myofibrils, myeloid structures, degenerated mitochondria, disarray of myofibrils, enlarged autophagic vacuoles and dilated sarcoplasmic reticulum vesicles were observed in the soleus of CERtreated rats (Figs. 4C to F).

Table 1. Histopathological properties of CER-induced myotoxicity (Experiment 2) 䇭 Group

Control

CER

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Day

Animal No.

CK activity (IU/l)

9 9 9 9 9 6 6 6 6 6 8 8 8 8 8 9

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

228 191 169 188 150 286 332 293 289 247 14810 692 411 143932 6852 2871

Soleus 䇭

䇭

䇭

䇭䇭

0 0 0 0 0 1 0 0 1 0 46 0 1 216 12 31

NADH-score Tibialis anterior 0 0 0 0 0 0 0 0 0 0 5 1 0 15 5 0

EDL 0 0 0 0 0 0 0 0 1 0 0 0 0 13 1 0


Fig. 2.

Findings of muscle fibers in CER-treated rats (Experiment 2). (A) Soleus and (B) tibialis anterior muscles of control animal on day 9, and (C) soleus and (D) tibialis anterior muscles of CER-treated rats on day 8. In the soleus muscle of the control rat (A), the muscle fibers are relatively uniform in size. In the soleus of the CER-treated rat (C), there is marked variation in fiber size with scattered dense fibers (arrow) and fibers with sarcoplasmic mass (arrowhead). There is no change between untreated (B) and treated (D) tibialis anterior muscles. A-D: hematoxylin and eosin staining. Bar = 100 μm.

Fig. 3.

NADH-TR staining of the muscle fibers. (A) Soleus and (B) tibialis anterior muscles of control rat on day 9, and (C) soleus and (D) tibialis anterior muscles of the CER-treated rat on day 8. In the soleus muscle of CER-treated rat (C), there is marked variation in fiber size and scattered densely stained fibers (arrow). There is no apparent difference in the tibialis anterior muscle between control (B) and CER-treated (D) rats. A-D: NADH-TR staining. Bar = 50 μm. Vol. 36 No. 4


Fig. 4.

Electron microscopic findings of the soleus. (A) Control rat on day 9, (B) CER-treated rat on day 6, (C, D, E and F) CERtreated rat on day 8. The following findings were noted: (A) no abnormalities; (B) swollen mitochondria and activated lysosomes (arrowheads), inclusion body in a mitochondrion (arrow) and autophagic vacuoles; (C) electron-dense mitochondria (arrowheads) and inclusion body in a mitochondrion (arrow); (D) autophagic vacuoles ingesting membranous organelles between the myofibrils (arrows) and myeloid structures (arrow head); (E) degenerated mitochondrion (arrow), activated lysosomes (arrowheads) and disarray of myofibril (dashed arrow); (F) enlarged autophagic vacuoles and dilated sarcoplasmic reticulum vesicles (asterisks). Bar = 1 μm.

DISCUSSION In the present study, abnormal findings in skeletal muscles were initially noted on Day 8 of CER treatment, including hyper-contracted (opaque) and variable in size and fiber necrosis, which is accompanying hind limb weakness and a marked elevation in plasma Vol. 36 No. 4

CK activity. These lesions were evident in the type I fiber-predominant soleus muscle, whereas cell injury was not evident in the type II fiber-predominant EDL or tibialis anterior muscles. NADH-TR staining implied abnormality in organelles in the type I muscular cell. Electron microscopic examination revealed that the mitochondrial changes including swollen mitochondria and activated


lysosomes were evident prior to the prevalence of injuries observed in the type I fiber muscle. At the early stage of CER-induced mitochondrial damage, mitochondria became round and electron dense, and then are rapidly degenerated and scavenged by lysosome. Following the mitochondrial damage, myofibrils were degenerated and numerous autophagic vacuoles were accumulated, resulting in the type I muscle fibers necrosis. These data suggest that mitochondria would be the primary target of the soleus-specific myotoxicity elicited by the CER treatment in the present study. Mitochondria are suggested to be a direct target of statin-induced toxicity both in vitro (De Pinieux et al., 1996) and in vitro (Kaufmann et al., 2006, Nishimoto et al., 2003), although severity of statin-induced mitochondrial toxicity highly depends on chemical property such as hydrophilicity (Kaufmann et al., 2006). Anatomically, the mitochondria were more abundant in the soleus compared with EDL (Gauthier, 1986), supporting a hypothesis that more abundant mitochondria in the soleus muscle, rather than in the EDL or tibialis anterior, may have been exaggerated the CER-induced myotoxicity. Such mitochondrial abnormalities in the statin-induced myotoxic lesions were consistently observed for other statins, including simvastatin, rosuvastatin, and pravastatin, in multiple species including humans (Bergman et al., 2003; Gambelli et al., 2004; Sirvent et al., 2005; Westwood et al., 2005; Seachrist et al., 2005; Westwood et al., 2008). Mitochondria are the organelles that show the first morphological changes following statin treatment, although there is controversy regarding whether mitochondria is the primary target of statin-induced myotoxicity in rats (Waclawik et al., 1993; Schaefer et al., 2004; Westwood et al., 2008). The type I fiber-targeted myotoxicity in the CERfed rats established in the present study is quite unique for statin-induced myopathy, because previous studies indicated that statin-induced myotoxicity majorly targets to type II fiber-predominant muscles. For example, Reijneveld et al. (1996) reported that the EDL was strongly damaged by the simvastatin treatment in 3-weekold male Wistar rats (Reijneveld et al., 1996), and other reports generally supports such type II fiber-targeted myotoxicity by statin treatment (Reijneveld et al., 1996; Westwood et al., 2005, 2008). Thus, the type I-specific myotoxic rat model established in our study will be unique and valuable tool to investigate mechanisms of statin-induced myotoxicity. Regarding the discrepancy of myotoxic target fiber type induced by statins between previous studies and ours, differences in the study designs, including age/strain of the rats, dosing period and statin compounds used, could have

affected considerable impacts on the myotoxic outcomes. In the previous studies, CER was reported to develop type II muscle fiber toxicity in several strains of the rats like Sprague-Dawley rats at 8-week-old (Schaefer et al., 2004) and Wistar rats at 6 to 8-week-old (Westwood et al., 2005). In addition, simvastatin induced type II muscle fiber toxicity in young male Wistar rats (i.e., 3-week-old) (Reijneveld et al., 1996). On the other hand, it is plausible that a myotoxic profile elicited by the statin treatment may be time-dependent: Westwood et al. (2005) observed a weak lesion in the soleus on days 5 and 6 of CER treatment (Westwood et al., 2005), but there was no lesion in the soleus any more on day 15, and instead severe injury was observed in the EDL (Westwood et al., 2005). Thus, it it is possible that CER may first have induced a cellular damage in the soleus, but the damage is quickly recovered and then EDL is more severely affected in the later time point, although more detailed time-course histopathological examination is needed to clarify this hypothesis. Interestingly, treadmill exercise, which leads to fiber-type switching from type II to type I fibers, was found to exacerbate the cerivastatin-induced myotoxicity (Seachrist et al., 2005), implying that type I fibers could be more sensitive to cerivastatin-induced myotoxicity under the certain experimental condition. Therefore, the type I fiberspecific myotoxic model established in the present study may give clues to address this phenomenon. In conclusion, we have generated a unique myotoxic animal model exhibiting the type I fiber-targeted myotoxicity, using young male F344 rat fed CER. Since there is no report of type I fiber-targeted myotoxic rat induced by statin so far, it will be a valuable animal model to gain insight into molecular mechanisms of statin-induced myotoxicity. Further investigation utilizing omics techniques (i.e., transcriptomics and metabolomics) may help us reveal detailed molecular mechanisms of statin-induced myotoxicity. REFERENCES Amelink, G.J. and Bär, P.R. (1986): Exercise-induced muscle protein leakage in the rat. Effects of hormonal manipulation. J. Neurol. Sci., 76, 61-68. Bergman, M., Salman, H., Djaldetti, M., Alexandrova, S., Punsky, I. and Bessler, H. (2003): Ultrastructure of mouse striated muscle fibers following pravastatin administration. J. Muscle Res. Cell. Motil., 24, 417-420. Biral, D., Ballarin, F., Toscano, I., Salviati, G., Yu, F., Larsson, L. and Betto, R. (1999): Gender- and thyroid hormone-related transitions of essential myosin light chain isoform expression in rat soleus muscle during ageing. Acta. Physiol. Scand., 167, 317323. De Pinieux, G., Chariot, P., Ammi-Saïd, M., Louarn, F., Lejonc,

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452 H. Obayashi et al. J.L., Astier, A., Jacotot, B. and Gherardi, R. (1996): Lipid-lowering drugs and mitochondrial function: effects of HMG-CoA reductase inhibitors on serum ubiquinone and blood lactate/pyruvate ratio. Br. J. Clin. Pharmacol., 42, 333-337. Degens, H., Yu, F., Li, X. and Larsson, L. (1998): Effects of age and gender on shortening velocity and myosin isoforms in single rat muscle fibres. Acta. Physiol. Scand., 163, 33-40. Farmer, J.A. (2001): Learning from the cerivastatin experience. Lancet, 358, 1383-1385. Gambelli, S., Dotti, M.T., Malandrini, A., Mondelli, M., Stromillo, M.L., Gaudiano, C. and Federico, A. (2004): Mitochondrial alterations in muscle biopsies of patients on statin therapy. J. Submicrosc. Cytol. Pathol., 36, 85-89. Gauthier, G.F. (1986): Skeletal muscle fiber types. In: Myology: basic and clinical (Engel, A.G. and Banker, B.Q., eds.), pp.255283, McGRAW-HILL BOOK COMPANY, Cleveland, Ohio. Kaufmann, P., Török, M., Zahno, A., Waldhauser, K.M., Brecht, K. and Krähenbühl, S. (2006): Toxicity of statins on rat skeletal muscle mitochondria. Cell. Mol. Life Sci., 63, 2415-2425. Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y. and Sugiyama, Y. (2003): Comparing myotoxic effects of squalene synthase inhibitor, T-91485, and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors in human myocytes. Biochem. Pharmacol., 66, 2133-2139. Pette, D. and Staron, R.S. (2000): Myosin isoforms, muscle fiber types, and transitions. Microsc. Res. Tech., 50, 500-509. Reijneveld, J.C., Koot, R.W., Bredman, J.J., Joles, J.A. and Bär, P.R. (1996): Differential effects of 3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors on the development of myopathy in young rats. Pediatr. Res., 39, 1028-1035. Riggs, J.E., Schochet, S.S.Jr., Kopitnik, T.A. and Gutmann, L. (1986): Target fibers in multiple sclerosis: implications for pathogenesis. Neurology, 36, 297-298. Rosenson, R.S. (2004): Current overview of statin-induced myopa-

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thy. Am. J. Med., 116, 408-416. Schaefer, W.H., Lawrence, J.W., Loughlin, A.F., Stoffregen, D.A., Mixson, L.A., Dean, D.C., Raab, C.E., Yu, N.X., Lankas, G.R. and Frederick, C.B. (2004): Evaluation of ubiquinone concentration and mitochondrial function relative to cerivastatin-induced skeletal myopathy in rats. Toxicol. Appl. Pharmacol., 194, 10-23. Seachrist, J.L., Loi, C.M., Evans, M.G., Criswell, K.A. and Rothwell, C.E. (2005): Roles of exercise and pharmacokinetics in cerivastatin-induced skeletal muscle toxicity. Toxicol. Sci., 88, 551-561. Sirvent, P., Bordenave, S., Vermaelen, M., Roels, B., Vassort, G., Mercier, J., Raynaud, E. and Lacampagne, A. (2005): Simvastatin induces impairment in skeletal muscle while heart is protected. Biochem. Biophys. Res. Commun., 338, 1426-1434. Smith, P.F., Eydelloth, R.S., Grossman, S.J., Stubbs, R.J., Schwartz, M.S., Germershausen, J.I., Vyas, K.P., Kari, P.H. and MacDonald, J.S. (1991): HMG-CoA reductase inhibitor-induced myopathy in the rat: cyclosporine A interaction and mechanism studies. J. Pharmacol. Exp. Ther., 257, 1225-1235. Staffa, J.A., Chang, J. and Green, L. (2002): Cerivastatin and reports of fatal rhabdomyolysis. N. Engl. J. Med., 346, 539-540. Thompson, P.D., Clarkson, P. and Karas, R.H. (2003): Statin-associated myopathy. JAMA, 289, 1681-1690. Waclawik, A.J., Lindal, S. and Engel, A.G. (1993): Experimental lovastatin myopathy. J. Neuropathol. Exp. Neurol., 52, 542-549. Westwood, F.R., Bigley, A., Randall, K., Marsden, A.M. and Scott, R.C. (2005): Statin-induced muscle necrosis in the rat: distribution, development, and fibre selectivity. Toxicol. Pathol., 33, 246-257. Westwood, F.R., Scott, R.C., Marsden, A.M., Bigley, A. and Randall, K. (2008): Rosuvastatin: characterization of induced myopathy in the rat. Toxicol. Pathol., 36, 345-352.