Simvastatin Treatment Does Not Ameliorate Muscle Pathophysiology in a Mouse Model for Duchenne Muscular Dystrophy

Abstract

Duchenne muscular dystrophy is an X-linked, recessive muscular dystrophy in which the absence of the dystrophin protein leads to fibrosis, inflammation and oxidative stress, resulting in loss of muscle tissue. Drug repurposing, i.e. using drugs already approved for other disorders, is attractive as it decreases development time. Recent studies suggested that simvastatin, a cholesterol lowering drug used for cardiovascular diseases, has beneficial effects on several parameters in mdx mice. To validate properly the effectiveness of simvastatin, two independent labs tested the effects of 12-week simvastatin treatment in either young (starting at 4 weeks of age) or adult (starting at 12 weeks of age) mdx mice. In neither study were benefits of simvastatin treatment observed on muscle function, histology or expression of genes involved in fibrosis, regeneration, oxidative stress and autophagy. Unexpectedly, although the treatment protocol was similar, simvastatin plasma levels were found to be much lower than observed in a previous study. In conclusion, in two laboratories, simvastatin did not ameliorate disease pathology in mdx mice, which could either be due to the ineffectiveness of simvastatin itself or due to the low simvastatin plasma levels following oral administration via the food.

Fig. 1

Four limb hanging test after 4 and 8 weeks of treatment for mice started simvastatin treatment at 12 weeks of age (n = 10–11). No significant differences were seen between the mdx controls and simvastatin treated mdx mice at either time.

Fig. 2

Muscle strength and membrane integrity after direct muscle stimulation. Force frequency and response to eccentric contractions in 24–week old mice after 12 weeks of simvastatin treatment. (A–B) Tibialis anterior (in situ). (C–D) Diaphragm, (in vitro) (n = 8–12). ^**p < 0.01, ^****p < 0.0001 compared to wild type mice.

Fig. 3

Fibrosis. Diaphragm of 24–week old mice after 12 weeks of simvastatin treatment. (A) hydroxyproline content. (B–C) expression of collagen type I (B) and III (C) (n = 10–12). ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared to wild type mice.

Fig. 4

Inflammation. Expression of (A) Lgals3 and (B) CD68 in the diaphragm of 24-week old mice after 12 weeks of simvastatin treatment (n = 10–12). ^*p < 0.05, ^***p < 0.001, ^****p < 0.0001 compared to wild type mice.

Fig. 5

De- and regeneration. Expression of different isoforms of myosin heavy chain in the diaphragm of 24–week old mice after 12 weeks of simvastatin treatment. (A) embryonic (Myh3). (B) neonatal (Myh8). (C) adult slow-twitch type Iβ (Myh7). (D–F) adult fast-twitch type IIx (Myh1) (D), IIa (Myh2) (E) and IIb (Myh4) (F) (n = 10–12). ^*p < 0.05, ^**p < 0.01, ^***p < 0.001, ^****p < 0.0001 compared to wild type mice.

Fig. 6

Autophagy. Expression of (A) atrogin–1 and (B) LC3B in the diaphragm of 24–week old mice after 12 weeks of simvastatin treatment (n = 10–12). ^**p < 0.01 compared to wild type mice.

Fig. 7

Oxidative stress. Expression of (A) Nox2 and (B) Nox4 in the diaphragm of 24–week old mice after 12 weeks of simvastatin treatment (n = 10–12). ^***p < 0.001, ^****p < 0.0001 compared to wild type mice.

Fig. 8

Nerve-stimulated muscle strength. Diaphragm of 16–week old mice after 12 weeks of simvastatin treatment. (A) Twitch contraction force. (B) Peak of the tetanic contraction force evoked by 7 s stimulation at 40 Hz. (C–D) Twitch (C) and tetanic (D) contraction force normalised to bodyweight. (E) Stimulation frequency-contraction relationship. Area-under-the-curve of 7 s stimulation at the indicated frequency (F) Peak tetanic contraction force during 7s of 40, 60, 80 and 100 Hz nerve stimulation. (G) Fatigue after 7 s of tetanic contraction at 40, 60, 80 and 100 Hz nerve stimulation, expressed as percentage of the peak force (n = 5–6). There are no significant differences between any of the groups.

Fig. 9

Fibrosis. Diaphragm of 16–week old mice after 12 weeks of simvastatin treatment. (A) Representative images of Sirius Red stained diaphragms (10x magnification) (B) quantification of collagen content by Sirius Red staining. (C–D) expression of (C) collagen I and (D) collagen III (n = 5–6). ^*p < 0.05, ^**p < 0.01 compared to wild type mice.

Fig. 10

Inflammation. Expression of (A) Lgals3 and (B) CD68 in the diaphragm of 16-week old mice after 12 weeks of simvastatin treatment (n = 5–6). ^*p < 0.05 compared to wild type mice.

Fig. 11

De- and regeneration. Expression of different isoforms of myosin heavy chain in the diaphragm of 16-week old mice after 12 weeks of simvastatin treatment. (A) embryonic (Myh3). (B) neonatal (Myh8). (C) adult slow-twitch type Iβ (Myh7). (D–F) adult fast-twitch type IIx (Myh1) (D), IIa (Myh2) (E) and IIb (Myh4) (F) (n = 5–6). ^*p < 0.05, ^**p < 0.01 compared to wild type mice.

Fig. 12

Autophagy. Expression of (A) atrogin–1 and (B) LC3B in the diaphragm of 16–week old mice after 12 weeks of simvastatin treatment (n = 5–6). There are no significant differences between any of the groups.

Fig. 13

Oxidative stress. 16–week old mice after 12 weeks of simvastatin treatment. (A–B) Gene expression of Nox2 (A) and Nox4 (B) in the diaphragm. (C–D) Expression of Nox2 protein in the tibialis anterior. Each lane represents an individual mouse (n = 5–6). ^*p < 0.05, ^**p < 0.01 compared to wild type mice.

Fig. 14

Simvastatin plasma levels. (A) At 30 min, 1 hour and 2.25 hour after single oral gavage (8 mg/kg) (n = 3–4). (B) After 2 week (AM/PM plasma collection) and 12 week treatment with 80 mg/kg simvastatin in the diet. (n = 5–8). ^*p < 0.05