The muscle-specific ubiquitin ligase atrogin-1/MAFbx mediates statin-induced muscle toxicity

Abstract

Statins inhibit HMG-CoA reductase, a key enzyme in cholesterol synthesis, and are widely used to treat hypercholesterolemia. These drugs can lead to a number of side effects in muscle, including muscle fiber breakdown; however, the mechanisms of muscle injury by statins are poorly understood. We report that lovastatin induced the expression of atrogin-1, a key gene involved in skeletal muscle atrophy, in humans with statin myopathy, in zebrafish embryos, and in vitro in murine skeletal muscle cells. In cultured mouse myotubes, atrogin-1 induction following lovastatin treatment was accompanied by distinct morphological changes, largely absent in atrogin-1 null cells. In zebrafish embryos, lovastatin promoted muscle fiber damage, an effect that was closely mimicked by knockdown of zebrafish HMG-CoA reductase. Moreover, atrogin-1 knockdown in zebrafish embryos prevented lovastatin-induced muscle injury. Finally, overexpression of PGC-1alpha, a transcriptional coactivator that induces mitochondrial biogenesis and protects against the development of muscle atrophy, dramatically prevented lovastatin-induced muscle damage and abrogated atrogin-1 induction both in fish and in cultured mouse myotubes. Collectively, our human, animal, and in vitro findings shed light on the molecular mechanism of statin-induced myopathy and suggest that atrogin-1 may be a critical mediator of the muscle damage induced by statins.

Figure 1. Atrogin-1 is induced in human biopsy samples from patients with statin-induced muscle injury.

Total RNA was extracted from human quadriceps muscle biopsies, and atrogin-1 mRNA was quantitated by real-time PCR as described in Methods. *P = 0.017, difference between groups by 1-way ANOVA.

Figure 2. Lovastatin causes reduction in myotube diameter.

C2C12 myotube morphology and mean diameter following treatment with lovastatin at various concentrations (A) or for various times (B). Control (0 μM) cultures were treated with reagent vehicle alone. Original magnification, ×100.

Figure 3. Lovastatin induces expression of both atrogin-1 mRNA and protein in cultured myotubes.

(A) Atrogin-1 mRNA expression was measured by real-time PCR in samples of total RNA extracted from C2C12 myotubes treated with 0, 1, 2.5, 5.0, and 10 μM lovastatin for 6 hours, 20 hours, and 36 hours, respectively. (B) C2C12 myotubes were treated with lovastatin for 48 hours at the indicated concentrations, protein lysates were prepared, and immunodetection using polyclonal anti–atrogin-1 antibody was performed as described in Methods. Atrogin-1 band intensity was quantitated by densitometry. Atrogin-1 expression induced by dexamethasone (5 μM) (45) was used as a positive control. (C) Protein degradation was measured as described in Methods. Rates are presented as the percentage increase from proteolytic rate in nontreated control cultures. Dexamethasone (10 μM) was used as a positive control.

Figure 4. Myotubes from atrogin-1 null (–/–) mice are resistant to lovastatin-induced damage.

(A) Atrogin-1 protein expression is absent in atrogin-1 null (–/–) myotubes. Myoblasts derived from atrogin-1 knockout mice (–/–) and corresponding wild type (+/+) littermates were differentiated into myotubes. Cultures were stimulated to express atrogin-1 with dexamethasone (5 μM) or infected with constitutively active FoxO3- or GFP-expressing adenovirus (45). Atrogin-1 expression was detected by Western blotting as in Figure 3. (B) Myotubes from atrogin-1 null (–/–) and wild-type (+/+) mice were treated with lovastatin at the indicated concentrations for 48 hours. Myotube morphology was examined, and diameter was measured. Original magnification, ×100.

Figure 5. Lovastatin treatment disrupts myofiber structure in zebrafish embryos.

(A) Zebrafish embryos (20 hpf) were treated with concentrations of lovastatin ranging from 0.025 to 5.0 μM for 12 hours. Embryos were fixed and stained with antimyosin heavy chain antibody (F59) as described in Methods. Representative somite phenotypes are shown. All panels are side views, anterior, left. Original magnification, ×200. (B) Quantitation of muscle damage. Morphological phenotypes shown in A were grouped into 3 classes: class 1 changes include bowing, gap formation, and blocked/disrupted fibers; class 2 changes include irregular fibers and diffuse appearance; class 3 changes are typified by irregular somite boundaries. Percentage of embryos displaying specific class defects as a function of lovastatin concentration are shown. Numbers of embryos quantitated are 151, 178, 163, 185, 189, and 180 for the lovastatin concentrations of 0, 0.025, 0.05, 0.5, 1.0, and 5 μM, respectively.

Figure 6. Targeted knockdown of zebrafish HMG-CoA reductase has a muscle phenotype similar to that with lovastatin treatment and can be rescued by zebrafish atrogin-1 knockdown.

(A) Myosin heavy chain staining of representative control, HMG-CoA reductase, and combined HMG-CoA reductase/atrogin-1 knockdown in zebrafish embryos. Original magnification, ×200. (B) Quantitation of muscle damage. Classes of morphological phenotypes are as described in Figure 5 legend. MS1 and MS2 are missense controls for morpholinos MO1 and MO2, respectively. Note that in each case, the injection of morpholinos against atrogin-1 almost completely abolishes the damage caused by HMG-CoA reductase knockdown. Number of embryos quantitated in each bar are 231, 182, 197, and 202 for control, and 220, 204, 240, and 215 for the atrogin-1 knockdown.

Figure 7. Atrogin-1 knockdown reduces lovastatin-induced muscle damage in zebrafish embryos.

Zebrafish embryos (20 hpf) were treated with lovastatin (0.5 μM) for 12 hours. Atrogin-1 mRNA was measured by PCR (A) and zebrafish atrogin-1 protein by Western blotting (B). Five embryos were used for each analysis. (C) Western blot probed for atrogin-1 using protein lysates (20 μg/lane) derived from control zebrafish embryos, zebrafish embryos injected with morpholino against atrogin-1, adult zebrafish muscle, adult zebrafish kidney, and adult zebrafish liver. Five embryos were used for each analysis. (D) Myosin heavy-chain staining of representative control and atrogin-1 knockdown embryos. Note that suppression of atrogin-1 protects from statin-induced damage. Original magnification, ×200. (E) Quantitation of muscle damage. Classes of morphological phenotypes are as described in Figure 5 legend. Note that at each lovastatin concentration, the injection of morpholinos against atrogin-1 almost completely abolishes the damage caused by lovastatin. Numbers of embryos quantitated are 235, 182, 197, and 202 for the controls and 220, 204, 240, and 215 for the atrogin-1 knockdowns at lovastatin concentrations of 0, 0.05, 0.5, and 1.0 μM, respectively. (F) Muscle fiber diameter was measured following myosin heavy-chain staining as described in Methods. At least 500 fibers were measured at each lovastatin concentration. Results were graphed as the ratio of mean experimental fiber size ± SEM/mean control fiber size ± SEM. Control fiber size: 7.60 ± 0.19 μM.

Figure 8. Lovastatin suppresses IGF-1 signaling.

(A) Lovastatin suppresses PI3K/AKT/FoxO signaling in C2C12 myotubes. C2C12 myotubes were treated for 24 hours with vehicle (v) or lovastatin at the indicated concentrations. Protein lysates were prepared and subjected to immunoblot analysis with various antibodies. (B) Lovastatin induces the atrogin-1 promoter in a FoxO-dependent manner in zebrafish embryos. Embryos at the 1-cell stage were injected with the 0.4-kb atrogin-1 promoter reporter with the FoxO sites present or mutated (45). Embryos were grown in the presence of 0.5 μM lovastatin and luciferase activity measured in embryo lysates 48 hours later. Constitutively active FoxO3a was coinjected as a positive control.

Figure 9. PGC-1α expression reduces lovastatin-induced atrogin-1 expression and muscle damage in zebrafish embryos.

(A) Myosin heavy chain staining of representative zebrafish embryo somites following injection of 100 pg PGC-1α cDNA or vehicle in the presence or absence of 0.5 μM lovastatin for 12 hours (left box) or morpholino oligonucleotides against z–PGC-1α (right box). Note that suppression of atrogin-1 protects from statin-induced damage and that suppression of PGC-1α has a similar muscle phenotype as lovastatin treatment. Original magnification, ×200. (B) Quantitation of muscle damage. Classes of morphological phenotypes are as described in Figure 5. Note that at each lovastatin concentration, the injection of PGC-1α cDNA almost completely abolishes the damage caused by lovastatin. Number of embryos quantitated are 137, 112, 139, and 122 for the controls and 120, 103, 108, and 107 for the PGC-1α–injected embryos at the lovastatin concentrations of 0, 0.05, 0.5, and 1.0 μM, respectively. (C) Western blot of atrogin-1 following 0.5 μM, 12 hours lovastatin treatment in zebrafish embryos injected or not with 100 pg PGC-1α cDNA. (D) Muscle fiber diameter was measured following myosin heavy chain staining as described in Methods in embryos injected with atrogin-1 morpholinos or PGC-1α cDNA. At least 500 fibers were measured at each lovastatin concentration. Results were graphed as the ratio of mean experimental fiber size ± SEM/mean control fiber size ± SEM. Control fiber size: 7.58 ± 0.10 μM. (E) Mitochondrial function is diminished by lovastatin treatment of zebrafish embryos. Cells from zebrafish embryos treated with varying concentrations of lovastatin (0–1 μM) were stained with MitoTracker, and fluorescence intensity, reflecting mitochondrial function, was detected by fluorescence-activated cell sorting. Representative data of mean fluorescence intensity from 3 independent experiments are shown. (F) PGC-1α augments mitochondrial staining and protects against lovastatin’s effects in zebrafish embryos. Embryos were injected with PGC-1α cDNA as described in Methods, then treated with lovastatin (0.5 μM) for 24 hours. As in E, dispersed embryonic cells were stained with MitoTracker and detected by FACS. Data are presented as percentage of mean fluorescence intensity in vehicle-treated embryos.

Figure 10. PGC-1α reduces lovastatin-induced atrogin-1 expression and muscle damage in C2C12 myotubes.

(A) C2C12 myotubes infected for 68 hours with control adenovirus or adenovirus bearing PGC-1α were visualized by GFP. 5 μM lovastatin or vehicle was present for the final 48 hours. Original magnification, ×100. (B) Western blot for atrogin-1 in control-infected or PGC-1α–infected C2C12 myotube cultures in the presence of increasing concentrations of lovastatin (0–5 μM) for 48 hours. Expression of mitochondrial electron transport proteins cytochrome oxidase IV and cytochrome c were monitored by immunoblot. Note that lovastatin-induced atrogin-1 expression in the presence of PGC-1α is suppressed.