Fluvastatin and Atorvastatin Affect Calcium Homeostasis of Rat Skeletal Muscle Fibers in Vivo and in Vitro by Impairing the Sarcoplasmic Reticulum/Mitochondria Ca -Release System

The mechanism by which the 3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors (statins) induce skeletal muscle injury is still under debate. By using fura-2 cytofluorimetry on intact extensor digitorum longus muscle fibers, here we provided the first evidence that 2 months in vivo chronic treatment of rats with fluvastatin (5 and 20 mg kg ) and atorvastatin (5 and 10 mg kg ) caused an alteration of calcium homeostasis. All treated animals showed a significant increase of resting cytosolic calcium [Ca ]i, up to 60% with the higher fluvastatin dose and up to 20% with the other treatments. The [Ca ]i rise induced by statin administration was not due to an increase of sarcolemmal permeability to calcium. Furthermore, the treatments reduced caffeine responsiveness. In vitro application of fluvastatin caused changes of [Ca ]i, resembling the effect obtained after the in vivo administration. Indeed, fluvastatin produced a shift of mechanical threshold for contraction toward negative potentials and an increase of resting [Ca ]i. By using ruthenium red and cyclosporine A, we determined the sequence of the statininduced Ca release mechanism. Mitochondria appeared as the cellular structure responsible for the earlier event leading to a subsequent large sarcoplasmic reticulum Ca release. In conclusion, we suggest that calcium homeostasis alteration may be a crucial event for myotoxicity induced by this widely used class of hypolipidemic drugs. Hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are the most useful agents for treatment of hypercholesterolemia. By blocking the rate-limiting step catalyzed by this enzyme in the mevalonate pathway, de novo synthesis of cholesterol is prevented, and low-density lipoprotein cholesterol uptake into cells is promoted (Goldstein and Brown, 1990). The different statins currently available (simvastatin, atorvastatin, lovastatin, pravastatin, fluvastatin, rosuvastatin) are generally well tolerated in patients. However, side effects may arise in skeletal muscle, ranging from transient increase in creatine kinase, muscle pain, and cramps to myositis and potentially lifethreatening rhabdomyolysis (Bellosta et al., 2004; Rosenson, 2004). Despite the numerous studies describing myopathy in animals and humans, the molecular mechanism of statininduced myotoxicity has not been completely elucidated. A variety of hypotheses have been formulated to explain such toxicity, including impairment of glycoprotein synthesis in the muscle membrane as well as reduction of ubiquinone concentration in mitochondria, causing severe deficits in energy metabolism (Evans and Rees, 2002). Interestingly, it was also proposed that an alteration of structures involved in Ca homeostasis could play a pivotal role in producing myocyte injury. After 2 to 3 months of chronic treatment of rats with simvastatin, the voltage threshold for contraction (mechanical threshold, MT), a calcium-sensitive index of excitation-contraction coupling, was shifted toward more negative potentials in fast-twitch muscle fibers (Pierno et al., 1999), an effect that is compatible with an increase of resting cytosolic calcium concentration ([Ca ]i). Moreover, in vitro studies showed a statin-induced elevation of [Ca ]i and suggested a possible interference of the drug with intracellular Ca stores (Nakahara et al., 1994; Inoue et al., 2003; Sirvent et al., 2005), which in turn might be responsible of cell damages via activation of Ca dependent proteolytic enzymes (Sacher et al., 2005). It is noteworthy that all statin-induced effects on skeletal muscle This study was supported by Italian “Ministero dell’Istruzione, dell’Università e della Ricerca” (FIRB RBAU015E9T) (to D.C.C.). Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.106.118331. ABBREVIATIONS: HMG-CoA, 3-hydroxy-methyl-glutaryl coenzyme A; EDL, extensor digitorum longus; [Ca ]i, intracellular calcium concentration; MT, mechanical threshold; gCl, resting chloride conductance; SR, sarcoplasmic reticulum; RyR, ryanodine receptor; PTP, permeability transition pore; RR, ruthenium red; CsA, cyclosporine A; SOCE, store-operated calcium entry; ANOVA, analysis of variance. 0022-3565/07/3212-626–634$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 321, No. 2 Copyright © 2007 by The American Society for Pharmacology and Experimental Therapeutics 118331/3199286 JPET 321:626–634, 2007 Printed in U.S.A. 626 at A PE T Jornals on July 9, 2017 jpet.asjournals.org D ow nladed from are strictly dependent on their lipophilicity, showing highly hydrophilic pravastatin, and no muscle toxicity, even at high doses (Nakahara et al., 1994; Pierno et al., 1995, 1999). Recently, a multidisciplinary study aimed to the identification of the cellular and tissue targets of a chronic treatment with newer powerful and lipophilic statins, such as fluvastatin and atorvastatin, on rat skeletal muscle was conducted (Pierno et al., 2006). Both statins resulted more potently than simvastatin in producing skeletal muscle injury. Indeed, although no histological changes were observed on muscle fibers, both statin treatments produced a significant negative shift of the potentials of MT at which fibers contracted. In addition, statins altered muscle excitability by reducing resting chloride conductance (gCl), the electrical parameter sustained by the voltage-gated chloride channel CLC-1 critical for the maintenance of membrane stability. Because CLC-1 is negatively regulated by the Ca -dependent protein kinase C (De Luca et al., 1998; Rosenbohm et al., 1999), the observed reduction of gCl supports the hypothesis that statin administration could effectively interfere with calcium-handling mechanism. Based on the above findings, we performed a 2-month in vivo administration with fluvastatin and atorvastatin to rats and evaluated ex vivo the effect of drug treatment on resting [Ca ]i by fura-2 cytofluorimetry on tendon to tendon intact fibers of extensor digitorum longus (EDL) muscle. In parallel, to further elucidate the statin-induced Ca release mechanism, we characterized the statin cellular effects after acute in vitro application. Our data showed that both statins caused a sustained increase in cytosolic Ca levels by interfering with intracellular stores, such as mitochondria and sarcoplasmic reticulum. Considering the crucial role of resting calcium ions in skeletal muscle function and maintenance, our findings contribute to a better understanding of the mechanism responsible for cramps, myalgia, and other muscular side effects induced by this widely used class of hypolipidemic drugs. Materials and Methods Animals Care and in Vivo Drug Treatment. Animal care and in vivo drug treatment were approved by the Italian Health Department and the Institutional Animal Care and Use Committee according to Italian law (DL 116/92) and the European Community Directive (86/609/EEC). Male Wistar rats (Charles River Laboratories, Calco Como, Italy) initially weighing 300 to 350 g were used. The animals were housed individually in rat appropriate cages and fed with a commercial rodent chow (approximately 30 g day , 4RF21; Charles River Laboratories) and tap water ad libitum. Rooms were maintained at a constant temperature (22–24°C) and exposed to a light cycle of 12 h day 1 (8:00 AM–8:00 PM). The animals were subdivided into six experimental groups as follows. The first group (nine rats) was chronically administered with 5 mg kg 1 day 1 fluvastatin, the second group (six rats) was administered with 20 mg kg 1 day 1 fluvastatin, the third group (six rats) was administered with 5 mg kg 1 day 1 atorvastatin, the fourth group (10 rats) was administered with 10 mg kg 1 day 1 atorvastatin, the fifth group (six rats) was administered with only the vehicle (aqueous methylcellulose, CMC) used to dissolve the drugs, and the sixth group (seven rats), an untreated control group. During the treatment period, all animals showed normal body weight gain and seemed to be in good health, with the exception of rats chronically treated with high doses of fluvastatin (20 mg kg ) (for details see Pierno et al., 2006). The doses of drugs were chosen based on data present in the literature regarding human and rodents, as reported previously (Pierno et al., 2006). Fluvastatin and atorvastatin dissolved in CMC (0.5%) suspension were administered orally via an esophageal cannula once a day for 2 months. For each rat, the weight-related dose was formulated so that the maximal volume of drug-containing suspension was 1 ml. Because the results obtained from the two control groups (untreated and CMC-treated rats) were similar, we have combined and showed data as unique control. In Vivo Study: Determination of the Forelimb Muscle Strength. The forelimb muscle force of control and treated rats was evaluated before and every week until the end of the treatment by means of a grip strength meter (Columbus Instruments, Columbus, OH). Five rats per group (five rats treated with 5 mg/kg fluvastatin, five rats treated with 20 mg/kg fluvastatin, five rats treated with 5 mg/kg atorvastatin, five rats treated with 10 mg/kg atorvastatin, and five control rats) were analyzed. For this measure, the rats were allowed to grasp a triangular ring connected to a force transducer and then gently pulled away until the grip was broken. The transducer saved the force value at this point as a measure of the maximal resistance; the animal can develop with its forelimbs (De Luca et al., 2003). Five measures (one to five) were taken from each animal within 3 min, and each value was normalized with respect to the weight of the animal. We calculated the difference between the fifth measure and the first measure in each animal as a muscle fatigability index. The mean values ( S.E.M.) of this difference of control and of treated rats were then analyzed for significance (by ANOVA test). Dissection of Native Muscle Fibers. For ex vivo and in vitro experiments, EDL muscles were removed from the animal under deep urethane anesthesia (1.2 g/kg body weight). Soon after the surgery, the rats that were still anesthetized were euthanized by anesthetic overdose. EDL muscles of the different groups of treated rats and of control rats were pinned in a dissecting dish containing 95% O2/5% CO2-gassed normal physiological solution at room temperature (22°C) for further dissection. Small bundles of 10 to 15 fibers arranged in a single layer were dissected lengthwise, tendon to tendon, with the use of microscissors, as described elsewhere (Fraysse et al., 2003). Fura-2 Fluorescence Measurements in Intact Muscle Fibers. Calcium measurements were performed using the membranepermeant Ca indicator fura-2 acetoxymethyl ester (Invitrogen, Pero, Milano, Italy). Loading of muscle fibers was performed for 2 h at 25°C in normal physiological solution containing 5 M fura-2 acetoxymethyl ester mixed to 0.05% (v/v) Pluronic F-127 (Invitrogen). After loading, muscle fibers were washed with normal physiological solution and mounted in a modified RC-27NE experimental chamber (Warner Instrument Inc., Hamden, CT) on the stage of an inverted Eclipse TE300 microscope (Nikon, Tokyo, Japan) with a 40 Plan-Fluor objective (Nikon). The mean sarcomere length was set to 2.5 to 2.7 m. Fluorescence measurements were made using a QuantiCell 900 integrated imaging system (Visitech International Ltd, Sunderland, UK) as described previously (Fraysse et al., 2003,