Inhibition of Rab1 GTPase and Endoplasmic Reticulum-to-Golgi Trafficking Underlies Statin's Toxicity in Rat Skeletal Myofibers

Abstract

HMG-CoA reductase inhibitor statins are used for the treatment of hypercholesterolemia. However, statins have adverse effects on skeletal muscles with unknown mechanism. We have reported previously that fluvastatin induced vacuolation and cell death in rat skeletal myofibers by depleting geranylgeranylpyrophosphate (GGPP) and suppressing small GTPases, particularly Rab (FASEB J 21:4087-4094, 2007). Rab1 is one of the most susceptible Rab isoforms to GGPP depletion and is essential for endoplasmic reticulum (ER)-to-Golgi trafficking. Here, we explored whether Rab1 and ER-to-Golgi vesicle trafficking were affected by statins in cultured single myofibers isolated from flexor digitorum brevis muscles of adult rats. Western blot analysis revealed that Rab1A protein resided predominantly in membrane but not in cytosol in control myofibers, whereas it was opposite in fluvastatin-treated myofibers, indicating that fluvastatin inhibited Rab1A translocation from cytosol to membrane. GGPP supplementation prevented the effect of fluvastatin on Rab1A translocation. Brefeldin A, a specific suppressor of ER-to-Golgi trafficking, induced vacuolation and cell death in myofibers in a manner similar to that of fluvastatin. Although ER-to-Golgi traffic suppression induces unfolded protein response (UPR) and cell death in some cell types, neither fluvastatin nor brefeldin A up-regulated UPR in myofibers. Immunofluorescence study revealed that the distribution of an ER marker, calnexin, was restricted to the region around nucleus with fluvastatin, suggesting the inhibition of ER membrane traffic by fluvastatin. We conclude that suppression of Rab1 GTPase and the subsequent inhibition of ER-to-Golgi traffic are involved in statin-induced skeletal myotoxicity.

Mechanism of Statin-Induced Rhabdomyolysis

“…One group using rat immortalized L6 myoblasts reported that the depletion of FPP is critical for myotoxicity (22), but another laboratory using the same cell line suggested that GGPP was the critical isoprenoid (12). In our hands, it was not FPP (3 mM), but GGPP (3 mM) alone, because the addition of GGPP, not FPP, in the presence of fluvastatin completely canceled the toxic effects of statins in skeletal myofibers (15,17). FPP also failed to rescue myotoxicity in a study using myotubes differentiated from C2C12 myoblasts (23).…”

“…We focused on Rab1, which was responsible for the traffic from the ER to the Golgi apparatus because this is the origin and the bottle-neck pathway for all the vesicle traffic systems. Application of 1 mM fluvastatin reduced the membrane-bound form of Rab1, and GGPP supplementation canceled the reduction of membrane-bound Rab1 as well as vacuolation and cell death (17). This directly indicates that fluvastatin treatment inactivated Rab1.…”

“…This directly indicates that fluvastatin treatment inactivated Rab1. Furthermore, the ER-Golgi traffic inhibitor brefeldin A also reproduced the effects of statins on myofibers (17). Therefore, statin-induced Rab1 inactivation must be an important step leading to the myotoxic effects of statins.…”

“…L6 skeletal myoblasts were also damaged by statins due to the inactivation of the Rho/ROCK system (12). However, the inhibitors of either Rho or ROCK did not damage myofibers, unlike (in contrast to their effects on) myoblasts (15,17). Furthermore, we tested the effect of GGTase inhibitors to determine whether geranylgeranylated small GTPases were involved in statin's adverse actions on myofibers.…”

“…GGTI-298 (N-[4-[2(R)-amino-3-mercaptopropyl]a mino-2-(1-naphthalenyl) benzoyl]-l-leucine methyl ester trifluoroacetate salt), a specific GGTase I inhibitor, did not reproduce the adverse effects of statins. On the other hand, perillyl alcohol, a selective GGTase II inhibitor, simulated vacuolation and cell death (15,17). Why were differentiated muscle cells insensitive to the inactivation of the Rho/ROCK system?…”