FASEB J. Uncover Your Biological Pathway
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published as doi: 10.1096/fj.07-8713com.
 (The FASEB Journal. 2007;21:4087-4094.)
© 2007 FASEB
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-8713comv1
21/14/4087    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakamoto, K.
Right arrow Articles by Kimura, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakamoto, K.
Right arrow Articles by Kimura, J.

Rab-small GTPases are involved in fluvastatin and pravastatin-induced vacuolation in rat skeletal myofibers

Kazuho Sakamoto*,1, Takashi Honda{dagger}, Sachihiko Yokoya{ddagger}, Satoshi Waguri and Junko Kimura*

Departments of
* Pharmacology,

{ddagger} Cell Science,

Anatomy and Histology, School of Medicine, and

{dagger} Department of Human Life Sciences, School of Nursing, Fukushima Medical University, Fukushima, Japan

1Correspondence: Department of Pharmacology, School of Medicine, Fukushima Medical University, Hikarigaoka 1, Fukushima 960-1295, Japan. E-mail: kazuho{at}fmu.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Three-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase inhibitors, known as statins, induce skeletal muscle injury including myalgia, myositis, and rhabdomyolysis. The mechanism of this myotoxicity remains unknown. This study examined the effect of statins on single skeletal myofibers enzymatically isolated from the rat flexor digitorum brevis muscles. Fluvastatin and pravastatin induced the formation of numerous vacuoles in the myofibers after 72 h of treatment. This effect progressed in a time- and concentration-dependent manner and, consequently, cell death occurred after 120 h. Electron micrographs revealed craters along the sarcolemma and swelling of the sarcoplasmic reticula and mitochondria, in addition to intracellular vacuoles. When caffeine was added after 72 h of fluvastatin treatment, contractile shortening of statin-treated myofibers was significantly attenuated and blebs formed on the surface of the myofibers. The coapplication of geranylgeranylpyrophosphate (GGPP) with fluvastatin prevented the morphological changes, while that of farnesylpyrophosphate (FPP) was ineffective. Furthermore, perillyl alcohol, an inhibitor of Rab geranylgeranyl transferase and geranylgeranyl transferase-I (GGTase-I), mimicked the effect of statins, while a specific GGTase-I inhibitor (GGTI-298) or a farnesyl transferase inhibitor (FTI-277) failed to do so. These results suggest that the inactivation of Rab GTPase, which involved in intracellular membrane transport, is a crucial factor in statin-induced-morphological abnormality in skeletal muscle fibers.—Sakamoto, K., Honda, T., Yokoya, S., Waguri, S., Kimura, J. Rab-small GTPases are involved in fluvastatin and pravastatin-induced vacuolation in rat skeletal myofibers.


Key Words: HMG-CoA reductase inhibitors • isoprenylation • myopathy • rhabdomyolysis • skeletal muscle


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
THREE-HYDROXY-3-METHYL-GLUTARYL-COA (HMG-CoA) reductase inhibitors called statins were developed as antihyperlipidemia drugs (1) Currently, statins are used by more than 76 million people around the world (2) . Besides lowering the blood cholesterol level, statins have various "pleiotropic effects" including antiinflammatory, antisclerotic, antiosteoporotic, and anticancer effects (3) . The pleiotropic effects are most likely due to statin-induced depletion of isoprenoides, because farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) are downstream products of mevalonate and essential isoprenoids for the activation of small GTPases including Ras, Rho, Rac, and Rab, which are the modulators of many physiological responses including the formation of cytoskeleton and intracellular vesicular transport (4) . However, statins also have adverse effects especially on skeletal muscles including myalgia, myositis, and rhabdomyolysis (5) . For example, cerivastatin was voluntarily withdrawn from the drug market because it killed 52 patients due to severe rhabdomyolysis (6 , 7) . Despite these serious adverse effects, the mechanism underlying the statin-induced myotoxicity remains unknown. Furthermore, there is no way to cure this disease, except by terminating the administration of statins.

In the early 1990s, a ubiquinone depletion hypothesis was proposed, because ubiquinone is an essential cofactor in mitochondrial respiration and is synthesized downstream from the mevalonate pathway. In fact, statins do decrease the plasma concentration of ubiquinone (8) . However, subsequent studies have argued against this hypothesis, because, although statins do decrease the serum ubiquinone concentration, there is no change in the skeletal muscle (9) nor did it change mitochondrial respiration level (10) . Furthermore, no correlation was proven between the ubiquinone concentration and statin myotoxicity (11) . In fact, ubiquinone supplementation had no effect on the risk of statin-induced myopathy (12 , 13) .

Statin-induced myotoxicity has been examined under in vitro conditions using cultured cell line. In the L6 skeletal myoblasts cell line, statin induced apoptosis due to inactivation of H-Ras (14 15 16) . However, in both in vivo and clinical studies others have demonstrated that statins induced vacuoles and degenerated organelles followed by necrosis (10 , 17 18 19 20) . Therefore, there is a discrepancy between the statin-induced toxicity in cultured cell lines and in in vivo conditions.

In the present study, the effects of statins were examined using single skeletal muscle fibers enzymatically isolated from rat flexor digitorum brevis (FDB) muscles. The single myofibers were cultured for up to 120 h, and statins were applied in the culture medium. The morphological changes of the myofibers were observed by using phase contrast microscopy, scanning electron microscopy (SEM), and transmitted electron microscopy (TEM). To elucidate the mechanism pharmacologically, isoprenoides, isoprenyl transferase antagonists, and protein kinase inhibitors were applied in the absence and presence of statins. Our results indicate, for the first time, that Rab GTPase is involved in statin-induced skeletal myopathy.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Preparation of myofibers
All experiments were performed in accordance with the regulation of the Animal Research Committee of Fukushima Medical University. The isolation of single skeletal myofibers was performed according to Bekoff and Betz (21) . Female rats (Wistar; 8- to 16-wk-old) were anesthetized with ether and exsanguinated. The total number of rats used in this study was 18. The body weight of the rats ranged 160–210 g. For anesthesia, an airtight glass container (2 l in volume) was filled with evaporated ether (from ~5 ml liquid ether absorbed in cotton), in which a rat was placed. The FDB muscles from both soles and connective tissues were removed under a binocular microscope (Nikon, Tokyo, Japan). The FDB muscles were cleaned and incubated for 2.5–3.0 h at 37°C in Ringer’s solution (2.7 mM KCl, 1.2 mM KH2PO4, 0.5 mM MgCl2, 137 mM NaCl, 8.1 mM NaHCO3, 1.0 mM CaCl2, and 10.0 mM glucose) containing 0.3% collagenase (Wako, Tokyo, Japan; lot# CER1568) and 0.001% penicillin-G (Banyu Pharmaceutical, Tokyo, Japan) and 0.001% streptomycin (Meiji Seika, Tokyo, Japan). After incubation, the muscles were washed four times with Ringer’s solution, and the fibers were dispersed in Dulbecco’s modified essential medium (DMEM) containing 10% fetal bovine serum (FBS) and 0.001% penicillin-G and 0.001% streptomycin using a fire-polished wide mouth Pasteur’s pipette. To decrease the number of free mononuclear cells (fibroblasts), which would eventually overgrow during culture, the preparation was sedimented once at 1 g. The sediments were transferred to a laminin-coated 12-well dish. If this procedure was omitted, then the shape of the myofibers could not be kept intact. The drugs were applied within 6 h of isolation.

Cell staining
The cell viability was examined using 0.2% trypan blue. The myofibers were added with trypan blue/PBS (Invitrogen, Carlsbad, CA, USA) for 5 min. Then the cells were washed three times with Ringer’s solution, and the number of trypan-blue stained cells and nonstained cells were counted. For the Oil Red-O staining, the myofibers in culture dishes were fixed in 4% buffered paraformaldehyde for 24 h followed by wash with PBS (2.6 mM KCl, 1.8 mM KH2PO4, 137 mM NaCl, and 8.1 mM Na2HPO4). The myfibers were placed in a 60% isopropyl alcohol solution for 1 s followed by a 1 h incubation in Oil Red-O dye. The samples were then briefly (1–3 s) rinsed with 60% isopropyl alcohol and washed for 3 min under running tap water.

Counting vacuoles per myofiber
Vacuoles, which appeared in each single isolated myofiber after statin-treatment, were counted using a phase contrast microscope (CK40; Olympus, Tokyo, Japan). The magnification was x400. By changing the focus, all vacuoles in the myofibers were counted. From 7 to 10 single myofibers were counted in every trial. To randomize the counting, the stage of the microscope was thus moved irregularly.

Cell counting and length measurement
Cells were counted using a phase contrast microscope (CK40; Olympus), and images were acquired with the attached digital camera system (DP11; Olympus). Myofiber length was measured using the printed images.

Electron microscopy
For SEM, myofibers attached to the laminin-coated coverslips were fixed with a solution containing 4% paraformaldehyde, 1% glutaralaldehyde, and 0.1 M cacodylate buffer (pH 7.2–7.4), dehydrated in graded alcohols, critical-point dried, and sputter-coated with 12–15 nm of gold. Myofibers were examined with JEM1210 (JEOL, Tokyo, Japan). For TEM, the fixed myofibers were treated with 1% osmium tetroxide in 0.1 M cacodylate buffer for 2 h at 4°C and dehydrated with an ascending series of ethanol, and pure acetone. The myofibers were then embedded with Epon 812 and kept in an oven at 80°C for 5 days. After polymerization, myofibers were sectioned using an ultramicrotome (MT-7000, RMC, Tucson, AZ, USA) into 70–90 nm sections. The sections were mounted on a copper mesh grid, dual stained with uranyl acetate and lead citrate, and examined with 1200 EX (JEOL, Tokyo, Japan) at 100 kV of an accelerating voltage.

Materials
Most pharmacological agents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Fluvastatin was a gift from Novartis (Basel, Switzerland). Rho kinase (ROCK) inhibitor Y27632 was provided by Welfide (Osaka, Japan). Pravastatin, Y27632, and exoenzyme C3 were dissolved in sterile PBS. GGPP and FPP were originally dissolved in methanol. Perillyl alcohol was diluted in ethanol. Other compounds were dissolved in dimethyl sulfoxide. The vehicle concentration was <0.1% and that had no significant effects.

Selection of statins
In this study, we used fluvastatin and pravastatin. Statins are chemically classified into an acid form (e.g., fluvastatin, pravastatin, cerivastatin, etc.) and a lactone form (e.g., lovastatin and simvastatin). Acid form statins have an HMG-CoA reductase inhibitory activity from the beginning, but the lactone forms are inactive until metabolized (22) . To see the direct effect of statins on cultured skeletal myofibers, the acid form of statin is better than the lactone form. Fluvastatin was readily available in our laboratory, where the effects of this drug on cardiac muscles and endothelial cells have been studied (23 24 25) . In addition, there is a report of fluvastatin-induced myotoxicity in an in vivo study using rat (26) . Pravastatin was used as a representative hydrophilic statin, because different pharmacokinetics have been suggested to exist between hydrophilic and lipophilic statins (27) . For example, the octanol water partition coefficient (Po/w), the typical index of lipophilicity, of pravastatin is 0.59 (28) , while that of fluvastatin is 40 times higher than that of pravastatin (29) .

Determination of concentrations of statins
The maximum serum level (Cmax) of fluvastatin reported in the clinical study was 190 ng/ml (=0.46 µM; ref. 30 ) and in basic animal studies 0.2–10 µM fluvastatin was used (23 24 25) . We referred to those concentrations in this study. For pravastatin, we compared the drug effect with that of fluvastatin and thus determined the concentration range.

Analysis
Each experiment was repeated 3–10 times. The data were expressed as the mean ± SE. Statistical significance between two groups or among multiple groups was evaluated using Student’s t test, Scheffé’s and Dunnet’s test after the F test or an one-way analysis of variants (ANOVA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Fluvastatin and pravastatin induced morphological abnormalities in skeletal myofibers
To examine the effect of statins on skeletal myofibers, isolated single myofibers were cultured in the absence or presence of statins. Figure 1 Aa shows a control myofiber cultured for 72 h after isolation. After incubation with 10 µM fluvastatin for 72 h, the myofibers developed numerous vacuoles (Fig. 1Ab ). After 120 h, the myofibers were swollen, the sarcomere structures disappeared, and blebs were extruding from the cell surface (Fig. 1Ad ). To quantify the morphological changes, the number of vacuoles in each myofiber was counted under a phase contrast microscopy and then the myofibers were stained with trypan blue to identify the number of dead cells. The average number of vacuoles was 0.7 ± 0.1 in a control myofiber and 77.0 ± 8.7 in myofibers treated with 10 µM fluvastatin (n=58 myofibers from 8 rats, P<0.01) for 72 h. After 120 h of fluvastatin treatment, the viability of myofibers was dramatically decreased to 29.3 ± 4.1% (Fig. 1D ) from 84.2 ± 1.3% of the control myofibers (n=5, P<0.01). The concentration and time dependency of morphological changes are summarized in Fig. 1B-D .


Figure 1
View larger version (25K):
[in this window]
[in a new window]

  		Figure 1. Statin-induced morphological changes in myofibers. A) Phase contrast micrographs. a) Control myofiber after 72 h in culture with fibroblasts in the background cells; b) Myofiber cultured with 10 µM fluvastatin (Flv) for 72 h; note background cells have disappeared. c) Control myofiber after 120 h in culture. d) Myofiber cultured with 10 µM fluvastatin for 120 h. Blebs are significant. B) Time-dependent changes in number of vacuoles per myofiber. Numbers of vacuoles was counted in 24–56 control fibers from 4–7 rats (open diamonds) or 24–56 fluvastatin-treated fibers (filled diamonds). **P < 0.01, compared with the control. C) Comparison of number of vacuoles induced by Flv and Prv. Myofibers were incubated with various concentrations of Flv (closed diamonds) or Prv (closed triangles) for 72 h. (**P<0.01, compared with the control). D) Time-dependent changes in percentage of trypan blue positive myofibers in control (open circles) and fluvastatin-treated fibers (filled circles; **P < 0.01, compared with control).

We also investigated pravastatin and found that pravastatin at concentrations higher than 10 µM showed morphological damage similar to those induced by fluvastatin (Fig. 1C ). From these results, the statin-induced myofiber damage could be divided into the following three stages: 1) The early stage: no morphological change (during 48 h with 10 µM fluvastatin). 2) The vacuolation stage: development of many vacuoles (after 72–96 h with 10 µM fluvastatin; Fig. 1Ab ). 3) The terminal stage: trypan blue positive myofibers with blebs (after 120 h with 10 µM fluvastatin; Fig. 1Ad ).

Ultrastructural changes in fluvastatin-treated myofibers
SEM and TEM were used to examine the statin-induced morphological changes in detail. SEM images revealed craters of various sizes on the surface of fluvastatin-treated myofibers (Fig. 2 B). The diameter of those craters ranged from 83 to 665 nm (302±18 nm average; 56 craters measured). Furthermore, TEM images revealed that empty vacuoles were lining along the sarcolemma (Fig. 3 B) and that mitochondria (Fig. 3C ) and sarcoplasmic reticula (SR) were swollen (Fig. 3B, D ), while myofilaments and transverse tubules (T-tubules) appeared to be intact (Fig. 3B-D ). In addition, the nucleus appeared to be invaded by multilayered organelles (Fig. 3C ) and vacuoles were also present in the perinuclear region (Fig. 3D ). Because vacuolation and the swellings of organelle are characteristic of statin-induced myopathy in both animal and human skeletal muscles (10 , 11 , 17 18 19 20) , the observation of similar results in single myofibers demonstrates that this culture system reproduced the in vivo results.


Figure 2
View larger version (124K):
[in this window]
[in a new window]

  		Figure 2. Scanning electronmicrographs of isolated skeletal myofiber. A) Control myofiber after 72 h of incubation. B) Myofiber cultured with 10 µM fluvastatin for 72 h.


Figure 3
View larger version (39K):
[in this window]
[in a new window]

  		Figure 3. Transmitted electron micrographs of isolated skeletal myofiber. A) Control myofibers and after 10 µM fluvastatin (B–D) for 72 h. Asterisks indicate vacuoles (B, C). Arrowheads indicate expanded SR (B, D); Triangles indicate mitochondrion (C); a circle indicates multilayered organelles (C); diamonds indicate nucleus (A, C, D); and inequality signs indicate T-tubules (B). Bars indicate 500 nm in A, D and 200 nm in B, C.

Identification of the isoprenoid responsible for fluvastatin-induced vacuolation
To identify the mechanism underlying the statin-induced morphological changes in myofibers, 3 µM GGPP or 3 µM FPP were first added to 10 µM fluvastatin to determine which isoprenoid was involved in the statin effect. The coapplication of GGPP with fluvastatin for 72 h significantly reduced the number of vacuoles (Fig. 4 Aa, B). The average number of vacuoles was 77.0 ± 8.7 in fibers incubated with fluvastatin only, while it was 0.7 ± 0.2 in those with fluvastatin and GGPP (P<0.01; n=34). FPP did not reduce the number of vacuoles (58.8±9.6; n=34, P>0.05; Fig. 4Aa, B ). These indicate that GGPP, but not FPP, is responsible for statin-induced vacuolation, suggesting that the inactivation of geranylgeranyl small GTPases are involved in the fluvastatin-induced vacuolation.


Figure 4
View larger version (40K):
[in this window]
[in a new window]

  		Figure 4. Effect of isoprenoides on statin-induced vacuolation in myofibers. A) Phase contrast micrographs. a) A myofiber cultured with 10 µM Flv plus 3 µM GGPP for 72 h. b) Myofibers cultured with 10 µM Flv plus 3 µM FPP for 72 h. B) Numbers of vacuoles averaged for a myofiber cultured for 72 h under control, with 10 µM Flv, with 10 µM Flv and 3 µM GGPP (Flv+GGPP), and with 10 µM fluvastatin and 3 µM FPP (Flv+FPP). **P < 0.01, compared with Flv.

Identification of the small GTPase responsible for fluvastatin-induced vacuolation
Among various small GTPases, Rab is known to play an important role in intracellular membrane transport (31) . Rab is a small GTPase prenylated only by GGPP and not by FPP (32) . Since GGPP, but not FPP, prevented the statin-induced vacuolation in the previous experiment, we hypothesized that Rab GTPases may be involved in this effect. Prenylation of different small GTPases is mediated by distinct isoprenyl transferases. Rab is primarily geranylgeranylated by a transferase called Rab geranylgeranyl transferase (Rab GGTase; ref. 32 ), which could be inhibited by perillyl alcohol (33) . If Rab is involved, then the effect of statins should be mimicked by perillyl alcohol but not by specific inhibitors of geranylgeranyl transferase-I (GGTase-I) or farnesyl transferase (FTase) (34) , which prenylates various small GTPases other than Rab (35) .

To our surprise, perillyl alcohol induced massive vacuolation in myofibers only in 3 h (Fig. 5 B). GGTI-298 (10 µM) and FTI-277 (1 µM) did not induce any morphological changes during the 9 h examined (Fig. 5B, C ). These results strongly indicate that Rab GTPases are involved in the statin-induced vacuolation.


Figure 5
View larger version (16K):
[in this window]
[in a new window]

  		Figure 5. Effects of various isoprenoid-transferase inhibitors on statin-induced vacuolation. A) Phase-contrast micrographs of myofibers cultured for 9 h with vehicle (ethanol 0.1%) (a), 1 mM perillyl alcohol (POH) (b), 10 µM GGTI-298 (GGTI) (c), and 1 µM FTI-277 (FTI) (d). B) Time-dependent changes in the number of vacuoles per myofiber. Myofibers were incubated in the absence (open circles) or presence with 1 mM POH (closed stars), 10 µM GGTI (open squares), and 1 µM FTI (closed squares). C) Number of vacuoles in each fiber were counted and averaged under various conditions including 0.3–1 mM POH which is Rab GGTase inhibitor; 10 µM GGTI, GGTase inhibitor; 1 µM FTI, FT inhibitor. Incubation was for 9 h. **P < 0.01, compared with control. D) Average number of vacuoles in control myofibers and those incubated with exoenzyme C3 (C3), 30 µM Y27432, or 10 µM wortomannin (Wort) for 72 h.

In addition, Rho specific inhibitor exoenzyme C3, Rho-kinase (ROCK) inhibitor Y27632, phosphatidylinositol 3-monophosphate kinase (PI3-K) inhibitor wortmannin, were also tested, because it has been shown that statin-induced inhibition of Rho/ROCK pathway and/or Ras/PI3-K pathway have been suggested for the statin-induced cytotoxicity (14 , 15 , 36) . However, in these myofibers, none of the above inhibitors reproduced the fluvastatin effect even after 72 h of application (Fig. 5D ), thus the involvement of either the Rho/ROCK or Ras/PI3-K pathways is unlikely.

To exclude the possibility that the statin-induced morphological changes were due to ubiquinone deficiency, muscle fibers were stained with Oil Red-O, because ubiquinone-deficient skeletal muscles developed lipid droplets which were sensitive to Oil Red-O staining (37) . The vacuoles were insensitive to Oil Red-O staining in this study (see Supplemental Figure), thus supporting the notion that ubiquinone was not involved in statin-induced vacuolation.

Effect of caffeine in statin-treated fibers
The functional changes in the myofibers treated with fluvastatin for 72 h (statin-fibers) were further investigated by applying caffeine to induce muscle contraction. The lengths of the myofibers before the administration of caffeine were similar in the control (749.7±12.6 µm, n=35 fibers isolated from 4 rats; Fig. 6 Aa, B) and statin-fibers (723.3±13.2 µm, n=45 fibers from 4 rats; P>0.05; Fig. 6Ab, B ). The application of 30 mM caffeine shortened the control fibers to final lengths of 190.6 ± 12.4 µm, (n=84 fibers from 4 rats) after 60 min (Fig. 6Ac, B ). In contrast, the caffeine-induced shortening of the statin-fibers was significantly less than that of the control and the final lengths were 437.9 ± 16.8 µm, (n=109 fibers from 4 rats; P<0.01 vs. control) at 60 min (Fig. 6Ad, B ). Interestingly, the statin-fibers developed many blebs after the application of caffeine (Fig. 6Ad, f; C ). The bleb formation was observed in 57.5 ± 12.2% (n=4, P<0.01 vs. control) of the statin-fibers, while it was seen in only 6.5 ± 4.9% (n=4) of the control fibers (Fig. 6Ac, e; C ). These observations suggest that the statin-fibers were thus functionally damaged, as well as morphologically.


Figure 6
View larger version (82K):
[in this window]
[in a new window]

  		Figure 6. Effect of 30 mM caffeine on control and fluvastatin-treated fibers. A) Phase-contrast micrographs of control myofibers cultured for 72 h in the absence (a, c, e) and presence (b, d, f) of 10 µM fluvastatin. a, b) Before caffeine application. c–f). At 30 min with caffeine. B) Time-dependent changes in lengths of control (open squares) myofibers and fluvastatin-treated (closed squares) myofibers on caffeine application. **P < 0.01, compared with control. C) Percentages of myofibers that developed blebs in control (open hexagons) and fluvastatin (closed hexagons). **P < 0.01, compared with control.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrated that fluvastatin and pravastatin induced vacuolation in single isolated rat skeletal myofibers after 72 h of application and the cell death occurred after 120 h. The development of vacuoles has been reported as characteristic morphological abnormality in statin-treated animal and human skeletal muscles (10 , 11 , 17 18 19 20) . Nakahara et al. (10) reported that vacuoles appeared just beneath the sarcolemma in rabbit by the administrations of simvastatin or pravastatin. In rats and mice, lovastatin and pravastatin induced the degeneration and swelling of organelles, including mitochondria and SR (18 , 19) . The electron micrographs in the present study revealed vacuoles, multilayered organelles, as well as swollen mitochondria and SR in myofibers, which are consistent with those studies. In this study, some of the vacuoles were located just beneath the sarcolemma (Fig. 3B ), and these account for the craters observed by the scanning electron microscopes (Fig. 2B ). This may occur when the vacuoles attached and penetrated the plasma membrane. This result implies that statin-induced morphological abnormality in myofibers is associated with the dysfunction of vesicular transport.

In humans, the therapeutic concentrations of fluvastatin (40 mg/day; oral dose) and pravastatin (40 mg/day; oral dose) correspond with the maximum serum level (Cmax) of 190 ng/ml (=0.46 µM) and 65 ng/ml (=0.15 µM), respectively (30 , 38) . In the present study using rat, the lowest observed effect levels (LOELs) of fluvastatin (1 µM) and pravastatin (10 µM) were only slightly higher than those Cmax values measured in humans. Another study using rat reported that when the Cmax value of pravastatin reached 2.2 µg/ml (=5.1 µM), vacuolation was observed in the skeletal muscle, but it was not observed at 0.56 µg/ml (=1.3 µM) (39) . Those values also correlate well with our data in Fig. 1C , which demonstrate that pravastatin induced the morphological abnormality at the concentration of 10 µM but not at 1 µM. These observations suggest that statin-induced myopathy was successfully reproduced in single isolated myofibers.

Cmax and the area under the concentration-time curve (AUC) of statins have been demonstrated to increase due to drug-drug interaction, liver dysfunction, and kidney dysfunction (40) . Especially, the coadministration of cyclosporin A increased Cmax and the AUC of pravastatin 7–8 times and 12 times higher, respectively, in humans (41) . In contrast, those of fluvastatin were only 2–3 times higher with cyclosporin A than with mono-administration (42) . This may explain the reason why the reported cases of rhabdomyolysis is greater for pravastatin than fluvastatin. Although the LOEL of pravastatin on the myofibers was higher than that of fluvastatin in this study, this may explain the reason why pravastatin clinically induced myotoxicity as well as other statins (43) .

The morphological characteristics observed in this study, including intracellular vacuolations and swellings of organelles, suggest that the statins may have disturbed the intracellular membrane vesicle transport in the myofibers. Rab small GTPases are essential for vesicular transport and thereby determine the shape of organelle (31) . More than 60 isoforms of Rab GTPases have been identified, and each plays a specific role at different intracellular events. For example, Rab1 is responsible for transportation of vesicles from the ER to the Golgi body and Rab8 carries newly synthesized transmembrane proteins from the Golgi body to the plasma membrane (31) . Geranylgeranylation is required for the activation of Rab GTPases (31 , 32) . Therefore, Rabs appear to be involved in the statin-induced vacuolation observed in myofibers.

Perillyl alcohol, which inactivates Rab by directly inhibiting Rab GGTase and prevents the geranylgeranylation of Rab, induced morphological changes similar to those with fluvastatin in myofibers. Although perillyl alcohol also inhibits GGTase-I (33) , a specific GGTase-I inhibitor GGTI-298 did not induce vacuolation in skeletal myofibers. This further indicates that Rabs are involved in the vacuolation of myofibers. The effect of perillyl alcohol appeared more rapidly than those of fluvastatin and pravastatin. This may be because the cells treated with fluvastatin had to deplete the endogenous GGPP before inactivating Rabs, while perillyl alcohol directly inhibited Rab GGTase and inactivated Rabs. In HeLa cells, lovastatin deformed the Golgi apparatus and ER by decreasing isoprenoid-binding to Rab GTPases (44) . However, the pathway by which the inactivation of Rabs leads to fatal cellular damage remains unclear. The inactivation of Rabs induces failure in vesicular trafficking, thus resulting in various harmful phenomena including termed as ER stress associated with the accumulation of unnecessary proteins (45 , 46) . In the present study, treatment with statins was lethal to myofibers after 120 h. According to the findings of recent reports, Rabs are more essential for cell survival than Ras or Rho in C. elegans germline cells and human lung epithelial cells (47) , and the overexpression of Rab1 rescues dopaminergic neurons from {alpha}-synuclein accumulation induced damage (48) .

Studies have demonstrated that simvastatin induces apoptosis, due to the depletion of FPP and Ras inactivation, thus resulting in PI3-K inhibition in the L6 skeletal myoblast cell line (14 , 15) . However, in the present study, FPP did not rescue myofibers from statin-induced morphological changes. Furthermore, specific FTase inhibitors, FTI-277 and wortmannin, did not induce any morphological changes in the myofibers. Therefore, Ras/PI3-K pathway is not involved in the statin-induced toxicity in skeletal myofibers. Taken together, there seems a difference in the statin effects between immortalized skeletal muscle cell line (49) and terminally developed myofibers freshly isolated from the skeletal muscle. Investigators should therefore be cautious when using such cell lines to analyze statin-induced myotoxicity.

These experiments confirmed that statin-induced myotoxicity was not due to ubiquinone depletion, because FPP, which is essential for ubiquinone biosynthesis, did not protect myofibers from statin-induced damage. Furthermore, Oil Red-O, a marker of ubiquinone deficiency, did not stain fluvastatin-induced vacuoles. The ragged red fibers and intramuscular vacuoles containing lipid droplets, which could be stained by Oil Red-O, are symptoms of ubiquinone deficiency. These structures are uniquely observed in type-I (slow/red/mitochondria-rich) muscles (37) . In contrast, statin-induced necrotic damage is specifically observed in type-II (fast/white/mitochondria-poor) muscles (19 , 50) . Therefore, the pathogenesis of statin-induced vacuolation and ubiquinone-deficient muscle disease is quite different. In fact, the FDB muscles from which the myofibers were isolated are composed of 90% type-II muscle fibers (51) . Analysis of the pathogenesis also suggests that antihyperlipidemia drugs that do not decrease GGPP, such as squalene synthase inhibitors (52) or squalene epoxidase inhibitors (53) , may be safer than statins for preventing skeletal muscle injury. Otherwise, the supplementation of statins with GGPP may be able to decrease the risk of statin-induced myopathy. We hope that this treatment modality may lead to the development of new antihyperlipidemia drugs that do not induce myotoxicity.

The caffeine-induced contractile shortening of statin fibers was significantly attenuated vs. that of the control. In addition, the statin fibers developed many blebs around the myofibers after caffeine application. These findings suggest that statin-induced morphological changes were accompanied by vulnerability to contractile stress in the sarcolemma of the myofibers. This result may account for the reports that physical exercise markedly increases the risk of the statin-induced myopathy (54 , 55) . In this study, we did not determine whether the statin-induced dysfunction of the skeletal muscle contractility is also related to the inactivation of Rab GTPases. Further study is necessary to clarify the mechanism of this phenomenon.

In the present study using isolated myofibers, hydrophilic pravastatin also induced significant morphological changes similar to that of fluvastatin but the potency was ~10-fold less than that of fluvastatin. Some reports revealed that pravastatin does not affect L6 myoblasts even in the milimolar range (56 , 57) . However, according to the findings of clinical studies, hydrophilic statins also have a risk of myotoxicity (5 , 43 , 58) . Thus, there is a clear difference between isolated myofibers and L6 myoblasts regarding the sensitivity of pravastatin. There is a possibility that the difference in the distribution of such drug transporters between myofibers and the other type of cells may account for the selectivity of the statins. This study is under way in our laboratory.

In conclusion, our findings suggest that statins induce vacuolation in skeletal myofibers by inactivating Rab small GTPases as a result of GGPP depletion. The morphological deformation by statins was accompanied by the functional vulnerability of the myofibers. Further study is necessary to identify which isoforms of Rab GTPases are involved in such statin-induced abnormality.


   ACKNOWLEDGMENTS
 
We thank S. Sato, H. Hu, T. Ono, H. Ikeda, S. Maeda, and Y. Shikama (Fukushima Medical University) for technical help. We also thank Ms. Kaori Kato (Nihon Medi-Physics, Sodegaura, Japan) for giving practical information of statin-induced myopathy. This work was supported by grants-in-aid from the Japan Foundation for Promotion of Science to J. Kimura (No. 17590223), from Fukushima Medical University to K. Sakamoto, and in part from the Smoking Research Foundation (KI18003) to J. Kimura.

Received for publication April 23, 2007. Accepted for publication June 14, 2007.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Endo, A. (1992) The discovery and development of HMG-CoA reductase inhibitors. J. Lipid Res. 33,1569-1582[Medline]
  2. Smith, C. C., Bernstein, L. I., Davis, R. B., Rind, D. M., Shmerling, R. H. (2003) Screening for statin-related toxicity: the yield of transaminase and creatine kinase measurements in a primary care setting. Arch. Intern. Med. 163,688-692[Abstract/Free Full Text]
  3. Liao, J. K., Laufs, U. (2005) Pleiotropic effects of statins. Annu. Rev. Pharmacol. Toxicol. 45,89-118[CrossRef][Medline]
  4. Takai, Y., Sasaki, T., Matozaki, T. (2001) Small GTP-binding proteins. Physiol. Rev. 81,153-208[Abstract/Free Full Text]
  5. Rosenson, R. S. (2004) Current overview of statin-induced myopathy. Am. J. Med. 116,408-416[CrossRef][Medline]
  6. Ucar, M., Mjorndal, T., Dahlqvist, R. (2000) HMG-CoA reductase inhibitors and myotoxicity. Drug. Saf. 22,441-457[CrossRef][Medline]
  7. Farmer, J. A. (2001) Learning from the cerivastatin experience. Lancet 358,1383-1385[CrossRef][Medline]
  8. Folkers, K., Langsjoen, P., Willis, R., Richardson, P., Xia, L. J., Ye, C. Q., Tamagawa, H. (1990) Lovastatin decreases coenzyme Q levels in humans. Proc. Natl. Acad. Sci. U. S. A. 87,8931-8934[Abstract/Free Full Text]
  9. Laaksonen, R., Jokelainen, K., Sahi, T., Tikkanen, M. J., Himberg, J. J. (1995) Decreases in serum ubiquinone concentrations do not result in reduced levels in muscle tissue during short-term simvastatin treatment in humans. Clin. Pharmacol. Ther. 57,62-66[CrossRef][Medline]
  10. Nakahara, K., Kuriyama, M., Sonoda, Y., Yoshidome, H., Nakagawa, H., Fujiyama, J., Higuchi, I., Osame, M. (1998) Myopathy induced by HMG-CoA reductase inhibitors in rabbits: a pathological, electrophysiological, and biochemical study. Toxicol. Appl. Pharmacol. 152,99-106[CrossRef][Medline]
  11. 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., 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[CrossRef][Medline]
  12. Hargreaves, I. P., Duncan, A. J., Heales, S. J., Land, J. M. (2005) The effect of HMG-CoA reductase inhibitors on coenzyme Q10: possible biochemical/clinical implications. Drug. Saf. 28,659-676[CrossRef][Medline]
  13. Levy, H. B., Kohlhaas, H. K. (2006) Considerations for supplementing with coenzyme Q10 during statin therapy. Ann. Pharmacother. 40,290-294[Abstract/Free Full Text]
  14. Nakagawa, H., Mutoh, T., Kumano, T., Kuriyama, M. (1998) HMG-CoA reductase inhibitor-induced L6 myoblast cell death: involvement of the phosphatidylinositol 3-kinase pathway. FEBS Lett. 438,289-292[CrossRef][Medline]
  15. Matzno, S., Yasuda, S., Juman, S., Yamamoto, Y., Nagareya-Ishida, N., Tazuya-Murayama, K., Nakabayashi, T., Matsuyama, K. (2005) Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho protein. J. Pharm. Pharmacol. 57,1475-1484[CrossRef][Medline]
  16. Dirks, A. J., Jones, K. M. (2006) Statin-induced apoptosis and skeletal myopathy. Am. J. Physiol. Cell. Physiol. 291,1208-1212[CrossRef]
  17. Chucrallah, A., De Girolami, U., Freeman, R., Federman, M. (1992) Lovastatin/gemfibrozil myopathy: a clinical, histochemical, and ultrastructural study. Eur. Neurol. 32,293-296[Medline]
  18. Waclawik, A. J., Lindal, S., Engel, A. G. (1993) Experimental lovastatin myopathy. J. Neuropathol. Exp. Neurol. 52,542-549[Medline]
  19. Westwood, F. R., Bigley, A., Randall, K., Marsden, A. M., Scott, R. C. (2005) Statin-induced muscle necrosis in the rat: distribution, development, and fibre selectivity. Toxicol. Pathol. 33,246-257[CrossRef][Medline]
  20. Bergman, M., Salman, H., Djaldetti, M., Alexandrova, S., Punsky, I., Bessler, H. (2003) Ultrastructure of mouse striated muscle fibers following pravastatin administration. J. Muscle. Res. Cell. Motil. 24,417-420[CrossRef][Medline]
  21. Bekoff, A., Betz, W. (1977) Physiological properties of dissociated muscle fibres obtained from innervated and denervated adult rat muscle. J. Physiol. 271,25-40[Abstract/Free Full Text]
  22. Greenspan, M. D., Yudkovitz, J. B., Alberts, A. W., Argenbright, L. S., Arison, B. H., Smith, J. L. (1988) Metabolism of lovastatin by rat and human liver microsomes in vitro. Drug. Metab. Dispos. 16,678-682[Abstract]
  23. Yokoyama, K., Ishibashi, T., Ohkawara, H., Kimura, J., Matsuoka, I., Sakamoto, T., Nagata, K., Sugimoto, K., Sakurada, S., Maruyama, Y. (2002) HMG-CoA reductase inhibitors suppress intracellular calcium mobilization and membrane current induced by lysophosphatidylcholine in endothelial cells. Circulation 105,962-967[Abstract/Free Full Text]
  24. Li, L., Matsuoka, I., Suzuki, Y., Watanabe, Y., Ishibashi, T., Yokoyama, K., Maruyama, Y., Kimura, J. (2002) Inhibitory effect of fluvastatin on lysophosphatidylcholine-induced nonselective cation current in Guinea pig ventricular myocytes. Mol. Pharmacol. 62,602-607[Abstract/Free Full Text]
  25. Maeda, S., Matsuoka, I., Iwamoto, T., Kurose, H., Kimura, J. (2005) Down-regulation of Na+/Ca2+ exchanger by fluvastatin in rat cardiomyoblast H9c2 cells: involvement of RhoB in Na+/Ca2+ exchanger mRNA stability. Mol. Pharmacol. 68,414-420[Abstract/Free Full Text]
  26. Matsuyama, K., Nakagawa, K., Nakai, A., Konishi, Y., Nishikata, M., Tanaka, H., Uchida, T. (2002) Evaluation of myopathy risk for HMG-CoA reductase inhibitors by urethane infusion method. Biol. Pharm. Bull. 25,346-350[CrossRef][Medline]
  27. Ichihara, K., Satoh, K. (2002) Disparity between angiographic regression and clinical event rates with hydrophobic statins. Lancet 359,2195-2198[CrossRef][Medline]
  28. Serajuddin, A. T., Ranadive, S. A., Mahoney, E. M. (1991) Relative lipophilicities, solubilities, and structure-pharmacological considerations of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors pravastatin, lovastatin, mevastatin, and simvastatin. J. Pharm. Sci. 80,830-834[CrossRef][Medline]
  29. Lindahl, A., Sandstrom, R., Ungell, A.L., Abrahamsson, B., Knutson, T.W., Knutson, L., Lennernas, H. (1996) Jejunal permeability and hepatic extraction of fluvastatin in humans. Clin. Pharmacol. Ther. 60,493-503[CrossRef][Medline]
  30. Kivisto, K. T., Kantola, T., Neuvonen, P. J. (1998) Different effects of itraconazole on the pharmacokinetics of fluvastatin and lovastatin. Br. J. Clin. Pharmacol. 46,49-53[CrossRef][Medline]
  31. Grosshans, B. L., Ortiz, D., Novick, P. (2006) Rabs and their effectors: achieving specificity in membrane traffic. Proc. Natl. Acad. Sci. U. S. A. 103,11821-11827[Abstract/Free Full Text]
  32. Leung, K. F., Baron, R., Seabra, M. C. (2006) Thematic review series: lipid posttranslational modifications. geranylgeranylation of Rab GTPases. J. Lipid Res. 47,467-475[Abstract/Free Full Text]
  33. Ren, Z., Elson, C. E., Gould, M. N. (1997) Inhibition of type I and type II geranylgeranyl-protein transferases by the monoterpene perillyl alcohol in NIH3T3 cells. Biochem. Pharmacol. 54,113-120[CrossRef][Medline]
  34. Lane, K. T., Beese, L. S. (2006) Thematic review series: lipid posttranslational modifications. Structural biology of protein farnesyltransferase and geranylgeranyltransferase type I. J. Lipid Res. 47,681-699[Abstract/Free Full Text]
  35. Goalstone, M. L., Leitner, J. W., Golovchenko, I., Stjernholm, M. R., Cormont, M., Le Marchand-Brustel, Y., Draznin, B. (1999) Insulin promotes phosphorylation and activation of geranylgeranyltransferase II. Studies with geranylgeranylation of rab-3 and rab-4. J. Biol. Chem. 274,2880-2884[Abstract/Free Full Text]
  36. Ogata, Y., Takahashi, M., Takeuchi, K., Ueno, S., Mano, H., Ookawara, S., Kobayashi, E., Ikeda, U., Shimada, K. (2002) Fluvastatin induces apoptosis in rat neonatal cardiac myocytes: a possible mechanism of statin-attenuated cardiac hypertrophy. Cardiovasc. Pharmacol. 40,907-915[CrossRef][Medline]
  37. Di Giovanni, S., Mirabella, M., Spinazzola, A., Crociani, P., Silvestri, G., Broccolini, A., Tonali, P., Di Mauro, S., Servidei, S. (2001) Coenzyme Q10 reverses pathological phenotype and reduces apoptosis in familial CoQ10 deficiency. Neurology 57,515-518[Abstract/Free Full Text]
  38. Kyrklund, C., Backman, J. T., Neuvonen, M., Neuvonen, P. J. (2004) Effect of rifampicin on pravastatin pharmacokinetics in healthy subjects. Br. J. Clin. Pharmacol. 57,181-187[CrossRef][Medline]
  39. Naba, H., Kuwayama, C., Kakinuma, C., Ohnishi, S., Ogihara, T. (2004) Eisai hyperbilirubinemic rat (EHBR) as an animal model affording high drug-exposure in toxicity studies on organic anions. Drug Metab. Pharmacokinet. 19,339-351[CrossRef][Medline]
  40. Shitara, Y., Sugiyama, Y. (2006) Pharmacokinetic and pharmacodynamic alterations of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors: drug-drug interactions and interindividual differences in transporter and metabolic enzyme functions. Pharmacol. Ther. 112,71-105[CrossRef][Medline]
  41. Park, J. W., Siekmeier, R., Merz, M., Krell, B., Harder, S., Marz, W., Seidel, D., Schuler, S., Gross, W. (2002) Pharmacokinetics of pravastatin in heart-transplant patients taking cyclosporin A. Int. J. Clin. Pharmacol. Ther. 40,439-450[Medline]
  42. Holdaas, H., Hagen, E., Asberg, A., Lund, K., Hartman, A., Vaidyanathan, S., Prasad, P., He, Y. L., Yeh, C. M., Bigler, H., et al (2006) Evaluation of the pharmacokinetic interaction between fluvastatin XL and cyclosporine in renal transplant recipients. Int. J. Clin. Pharmacol. Ther. 44,163-171[Medline]
  43. Omar, M. A., Wilson, J. P. (2002) FDA adverse event reports on statin-associated rhabdomyolysis. Ann. Pharmacother. 36,288-295[Abstract]
  44. Ivessa, N. E., Gravotta, D., De Lemos-Chiarandini, C., Kreibich, G. (1997) Functional protein prenylation is required for the brefeldin A-dependent retrograde transport from the Golgi apparatus to the endoplasmic reticulum. J. Biol. Chem. 272,20828-20834[Abstract/Free Full Text]
  45. Xu, C., Bailly-Maitre, B., Reed, J. C. (2005) Endoplasmic reticulum stress: cell life and death decisions. J. Clin. Invest. 115,2656-2664[CrossRef][Medline]
  46. Komatsu, M., Waguri, S., Chiba, T., Murata, S., Iwata, J., Tanida, I., Ueno, T., Koike, M., Uchiyama, Y., Kominami, E., Tanaka, K. (2006) Loss of autophagy in the central nervous system causes neurodegeneration in mice. Nature 441,880-884[CrossRef][Medline]
  47. Lackner, M., Kindt, R., Carroll, P., Brown, K., Cancilla, M., Chen, C., de Silva, H., Franke, Y., Guan, B., Heuer, T., et al (2005) Chemical genetics identifies Rab geranylgeranyl transferase as an apoptotic target of farnesyl transferase inhibitors. Cancer Cell 7,325-336[CrossRef][Medline]
  48. Cooper, A. A., Gitler, A. D., Cashikar, A., Haynes, C. M., Hill, K. J., Bhullar, B., Liu, K., Xu, K., Strathearn, K. E., Liu, F., et al (2006) Alpha-synuclein blocks ER-Golgi traffic and Rab1 rescues neuron loss in Parkinson’s models. Science 313,324-328[Abstract/Free Full Text]
  49. Yaffe, D. (1968) Retention of differentiation potentialities during prolonged cultivation of myogenic cells. Proc. Natl. Acad. Sci. U. S. A. 61,477-483[Free Full Text]
  50. Smith, P. F., Eydelloth, R. S., Grossman, S. J., Stubbs, R. J., Schwartz, M. S., Germershausen, J. I., Vyas, K. P., Kari, P. H., 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[Abstract/Free Full Text]
  51. Carlsen, R. C., Larson, D. B., Walsh, D. A. (1985) A fast-twitch oxidative-glycolytic muscle with a robust inward calcium current. Can. J. Physiol. Pharmacol. 63,958-965[Medline]
  52. Nishimoto, T., Tozawa, R., Amano, Y., Wada, T., Imura, Y., 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[CrossRef][Medline]
  53. Matzno, S., Yamauchi, T., Gohda, M., Ishida, N., Katsuura, K., Hanasaki, Y., Tokunaga, T., Itoh, H., Nakamura, N. (1997) Inhibition of cholesterol biosynthesis by squalene epoxidase inhibitor avoids apoptotic cell death in L6 myoblasts. J. Lipid Res. 38,1639-1648[Abstract]
  54. Thompson, P. D., Zmuda, J. M., Domalik, L. J., Zimet, R. J., Staggers, J., Guyton, J. R. (1997) Lovastatin increases exercise-induced skeletal muscle injury. Metabolism 46,1206-1210[CrossRef][Medline]
  55. Seachrist, J. L., Loi, C. M., Evans, M. G., Criswell, K. A., Rothwell, C. E. (2005) Roles of exercise and pharmacokinetics in cerivastatin-induced skeletal muscle toxicity. Toxicol. Sci. 88,551-561[Abstract/Free Full Text]
  56. Nakahara, K., Yada, T., Kuriyama, M., Osame, M. (1994) Cytosolic Ca2+ increase and cell damage in L6 rat myoblasts by HMG-CoA reductase inhibitors. Biochem. Biophys. Res. Commun. 202,1579-1585[CrossRef][Medline]
  57. Kaufmann, P., Torok, M., Zahno, A., Waldhauser, K. M., Brecht, K., Krahenbuhl, S. (2006) Toxicity of statins on rat skeletal muscle mitochondria. Cell. Mol. Life. Sci. 63,2415-2425[CrossRef][Medline]
  58. Alsheikh-Ali, A. A., Ambrose, M. S., Kuvin, J. T., Karas, R. H. (2005) The safety of rosuvastatin as used in common clinical practice: a postmarketing analysis. Circulation 111,3051-3057[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 ANN INTERN MEDHome page
T. R. Joy and R. A. Hegele
Narrative Review: Statin-Related Myopathy
Ann Intern Med, June 16, 2009; 150(12): 858 - 868.
[Abstract] [Full Text] [PDF]

Home page
 Mayo Clin Proc.Home page
B. B. Murinson and N. J. Maragakis
Fluvastatin, Rhabdomyolysis, and Myotoxicity
Mayo Clin. Proc., November 1, 2008; 83(11): 1296 - 1296.
[Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
fj.07-8713comv1
21/14/4087    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Sakamoto, K.
Right arrow Articles by Kimura, J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sakamoto, K.
Right arrow Articles by Kimura, J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS