Skeletal Muscle Susceptibility to Clofibrate Induction of Lesions in Rats

Abstract

Morphological changes induced by clofibrate in type-1 predominant soleus, type-2 predominant tensor fasciae latae, and type-1 and -2 mixed biceps femoris muscles and diaphragm in rats were investigated. Administration of the agent at 500 or 750 mg/kg/day by oral gavage for 14 or 28 days caused lesions in the soleus muscle and diaphragm, bur no changes in the tensor fasciae latae and biceps femoris muscles. In soleus muscle, vacuolation of muscle fibers was observed in all animals treated with clofibrate, and degeneration of muscle fibers and infiltration of leukocytes were noted at 750 mg/kg/day. In diaphragm, vacuolation of muscle fibers was also observed in all animals treated with clofibrate, and these lesions were located in type-1 skeletal muscles densely stained with NADH-TR. The vacuoles seen in soleus muscle and diaphragm were positive for oil red O staining. In addition, increase of lipid droplets and mitochondrial hypertrophy was seen in soleus muscle, ultrastructurally. These data suggest that sensitivity to clofibrate-induced muscle toxicity differs among muscles, with type-1 fibers being susceptible.

Introduction

Hyperlipidemia is a major risk factor for cardio- and cerebrovascular disease and therefore hypolipidemic drugs such as fibrates and statins have found wide application. These are generally considered safe and well tolerated (Johnson et al., 2005).

Fibrates are effective at lowering elevated plasma triglycerides by activation of the nuclear receptor peroxisome proliferator-activated receptor-α (PPARα) in hepatocytes (Staels et al., 1998; Brunmair et al., 2004; Matzno et al., 2006), inducing expression of genes involved in intracellular fatty acid metabolism, such as mitochondrial β-oxidation (Staels et al., 1998; Matzno et al., 2006). However, they may also cause myopathy and rhabdomyolysis in humans (Lane and Mastaglia, 1978; Hodel, 2002; Warren et al., 2002 ).

HMG-CoA reductase inhibitors, statins, are also assosicated with skeletal myopathy (Evans and Rees 2002a, 2002b; Hodel, 2002; Thompson et al., 2003). Schaefer et al. (2004) previously reported induction skeletal myopathy in type-2 muscle fibers of rats by cerivastatin. They also described mitochondrial alteration, including disorganized cristae, flocculent matrix material, and membranous whorls in myofibers with degeneration. However, only few details has been reported regarding the myopathy induced by fibrates in animals (Teravainen et al., 1977; Afifi et al., 1984).

In the present study, we therefore investigated morphological changes in rat skeletal muscles induced by clofibrate histologically and ultrastructurally. For this purpose, type-1 predominant soleus, type-2 predominant tensor fasciae latae, and type-1 and -2 mixed biceps femoris muscles and diaphragm were selected for examination.

Materials and Methods

Animals

Female Crl:CD(SD) rats aged 5 weeks were purchased from Charles River Laboratories Japan, Inc. (Yokohama, Japan) and acclimatized for 1 week in an air-conditioned animal room at 22°C with a 12-hour light/dark cycle. The animals were given CRF-1 (Oriental Yeast Co., Ltd, Tokyo, Japan) and tap water ad libitum.

Dosing

Three rats were treated with clofibrate (Wako Pure Chemical Industries, Ltd., Osaka, Japan) at 500 mg/kg (b.i.d.) by oral gavage for 14 days. Six further rats received clofibrate at 750 mg/kg (once daily) for 28 days, with half of the animals sacrificed on Day 15. Clofibrate was suspended at 100 mg/mL or 150 mg/mL in a 0.5% carboxymethyl cellulose sodium salt (CMC-Na, Sigma-Aldrich Japan, Tokyo, Japan). Three additional rats were given 0.5% CMC-Na for 28 days as controls.

Necropsy and Tissue Collection

On Day 15 and 29, rats were sacrificed by exsanguinations by cutting the abdominal aorta under ether anesthesia. Soleus, tensor fasciae latae and biceps femoris muscles of both hindlimbs, as well as the diaphragm were removed. Muscles of the right hindlimb and right side of the diaphragm were frozen immediately in liquid nitrogen and those of left hindlimb and diaphragm were fixed in 10% neutral-buffered formalin. Soleus and tensor fasciae latae muscles of left hindlimb from rats treated with 750 mg/kg/day clofibrate for 28 days and control animals were also fixed in 2.5% glutaraldehyde in phosphate buffer for electron microscopy.

Light Microscopy

Frozen muscle was sectioned in 10-μm thickness using a cryostat maintained at −23°C. Serial sections were stained with hematoxylin and eosin (HE) and for reduced nicotinamide adenine dinucleotide-tetrazolium reductase (NADH-TR) to classify muscle fibers. All muscles from rats treated with clofibrate at 750 mg/kg/day for 28 days and control animals were also stained with oil red O for the identification of triglyceride. Formalin-fixed muscles were routinely embedded in paraffin, sectioned, and stained with HE.

Electron Microscopy

Soleus and tensor fasciae latae muscles from representative rats were postfixed in 1% osmium tetroxide and processed to epoxy resin blocks for electron microscopic examination. Semithin sections from each block, stained with toluidine blue, were used to select areas for electron microscopic examination. Ultrathin sections were stained with uranyl acetate and lead citrate, and examined under a Hitachi H-7500 electron microscope.

All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee of Mitsubishi Pharma Corporation.

Results

Microscopically, clofibrate-related changes with vacuolation of muscle fibers were noted in the soleus muscle and diaphragm of all treated animals (Figure 1). In contrast, no morphological alterations were apparent tensor fasciae latae and biceps femoris muscles. In the diaphragm, the lesions were located in type-1 skeletal muscles densely stained with NADH-TR (Figure 2). The vacuoles seen in soleus muscle and diaphragm were positive for oil red O staining (Figure 2).

Additionally, in the soleus muscle, degeneration of muscle fibers and infiltration of leukocytes were noted in rats treated with clofibrate at 750 mg/kg/day for 14 days or 28 days (Figure 3). Myofiber vacuolation was widespread in the soleus and diaphragm, whereas degenerative myofibers were focal and affected lesser numbers of myofibers and only in the soleus.

Ultrastructurally, increase of lipid droplets and hypertrophy of mitochondria were also seen in the soleus muscle (Figure 4). The microscopic change of muscular vacuolation corresponded with increase of lipid droplets.

Discussion

In the present study, clofibrate was found to induce lesions limited to type-1 predominant soleus muscle, and type-1 muscle fibers in diaphragm of rats. Degeneration, increase of lipid droplets, and mitochondrial hypertrophy were observed. However, in type-2 predominant tensor fasciae latae muscle, and type-2 muscle fibers in diaphragm, no morphological changes were observed. These data clearly indicate that the sensitivity to clofibrate-induced muscle toxicity differs among the muscles. In contrast, one of the statins, cerivastatin, is known to induce skeletal myopathy involving type-2 muscle fibers of rats (Schaefer et al., 2004). From these data, the mechanisms of muscle toxicity caused by fibrates and statins may differ from each other.

The target muscle fibers may offer clues in this regard. Clofibrate-induced myopathy located in type-1 muscle fibers may be related to the drug efficacy and its effects on PPAR α expression. PPAR α is predominantly found in tissues that metabolize high amounts of fatty acids, such as liver, kidney, heart, and muscles (Staels et al., 1998). Russell et al. (2003) described PPAR α protein content to be higher in type-1 fibers than in their type-2 counterparts.

In the present study, mitochondrial hypertrophy and increase of lipid droplet were noted in soleus muscle. Similar lesions have been described in carnitine deficiency and zidovudine-induced myopathy in humans, these muscular disorders being considered correlated with mitochondrial impairment (Boudin et al., 1976; Dalakas et al., 1994; Mascaro et al., 1998). It has been reported that fibrates are effective in lowering elevated plasma triglycerides, but also induces mitochondrial dysfunction (Brunmair et al., 2004). Therefore, mitochondrial dysfunction might be one of causes of clofibrate-induced myopathy.

Recently, it was reported that clofibrate-induced myopathy is related to carcium influx (Ikemoto and Endo, 2001; Matzno et al., 2006). Hodel briefly reviewed clofibrate-induced myopathy in rats (Hodel, 2002) but little concrete evidence has hitherto been available (Teravainen et al., 1977; Afifi et al., 1984).