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

Figures

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.

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  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.