Reducing sarcolipin expression mitigates Duchenne muscular dystrophy and associated cardiomyopathy in mice

Figures

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  Fig. 1. Reduction in SLN expression prolongs the lifespan of mdx:utr −/− mice. a The body weight of mdx:utr −/− :sln +/− and tKO mice in comparison to that of age- and sex- matched WT and mdx:utr −/− control mice. We used 3–4 month old mice for this experiment. The n number for each group is shown within the bar. Data are presented as mean ± SEM. **significantly different from other groups (p < 0.005, t-test with Welch’s correction). b Kaplan–Meier survival curves indicate that the mdx:utr −/− :sln +/− and tKO mice has increased lifespan in comparison to that of mdx:utr −/− controls as determined by the nonparametric log-rank test; p < 0.0001. c Increased Ca2+ dependent SR Ca2+ uptake, d increased V max of Ca2+ uptake and e decreased EC50 values in the diaphragm of mdx:utr −/− :sln +/− and tKO mice indicates improved SERCA function. The n number for each group is shown within the bar. Data are presented as mean ± SEM. **significantly different from other groups (p < 0.0005, t-test with Welch’s correction). *significantly different from WT and mdx:utr −/− mice (p < 0.005, t-test with Welch’s correction). f–h Representative western blot analysis and quantitation of SLN, SERCA1, SERCA2a and…

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  Fig. 2. Reduction in SLN expression ameliorates muscle pathology. a Calpain activity is restored to normal levels in the pectoral muscles of mdx:utr −/− :sln +/− and tKO mice. Data are presented as mean ± SEM (t-test with Welch’s correction) of five independent experiments performed in duplicates. The n number for each group is shown within the bar. b Representative H&E and Masson’s trichrome stained quadriceps and diaphragm muscles. Arrow indicates increased mononuclear infiltration (indicative of necrosis) and collagen (blue) accumulation (indicative of fibrosis) in mdx:utr −/− mice. Original magnification is ×20. Scale bar=100 μm. c–f Quantitation show that the necrotic and fibrotic areas were significantly reduced in both diaphragm and quadriceps of mdx:utr −/− :sln +/− mice and in the quadriceps of tKO mice in comparison to that of mdx:utr −/− controls. The n number for each group and the p values (t-test with Welch’s correction) are shown within the graph. Data are presented as mean ± SEM. Tissues from 3 to 4 month old mice are used for all the above experiments

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  Fig. 3. Reduction in SLN expression improves muscle regeneration and prevents fiber-type transition. a Representative images of WGA stained sections of quadriceps used for fiber size measurements. Original magnification is ×10. The measurements of minimal “Feret’s” diameter variance coefficients (VC) of the muscle fiber size shown in the bar graph indicates that VC of fiber size is significantly reduced in the quadriceps of mdx:utr −/− :sln +/− and tKO mice. Data are presented as mean ± SEM of four independent experiments. *significantly different from other groups (p < 0.05, t-test with Welch’s correction). Scale bar=100 μm. b Representative images of pectoral muscle sections immunostained for eMyHC (green) or Type I MyHC (green) and stained with WGA (red). Original magnification is ×10. Scale bar=100 μm. The quantitation of muscle fibers positive for eMyHC and Type I MyHC are shown in the bar graph. The n number for each group and the p values (t-test with Welch’s correction) are shown within the graph. Data are presented as mean ± SEM. NS-not statistically significant. Tissues from 3 to 4 month old mice are used for all the above experiments

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  Fig. 4. Reduction in SLN expression improves skeletal muscle function in mdx:utr −/− mice. a Forelimb strength measured using a grip strength meter shows improved muscle strength in the mdx:utr −/− :sln +/− and tKO mice. We used 3–4 month old male and female mice for this study. The n number for each group is shown within the bar. Data are presented as mean ± SEM (t-test with Welch’s correction). # p < 0.0001 vs. mdx:utr −/−. *p < 0.05 vs. mdx:utr −/− :sln +/− and tKO mice. b, e Representative traces of twitch force at 2 Hz for EDL and hemidiaphragm respectively. c, f The maximum force generated by the EDL and hemidiaphragm at 2 Hz are significantly increased in the mdx:utr −/− :sln +/− mice compared to that of mdx:utr −/− mice. The n number for each group is shown within the bar. Data are presented as mean ± SEM. d, g Force-frequency curves indicating that force generated by EDL and hemidiaphragm in the mdx:utr −/− :sln +/− mice are significantly increased at all frequencies. EDL and hemidiaphragm are from 3 to 4 month old mice

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  Fig. 5. Ablation of SLN expression prevents cardiomyopathy in mdx:utr −/− mice. All cardiac studies are performed in 3–4 month old mice. a Representative H&E and Masson’s trichrome stained ventricular tissue sections. Original magnification is ×10. Scale bar=100 μm. Bar graph indicate that reduction or ablation of SLN significantly reduced fibrosis and necrosis in the mdx:utr −/− hearts. The n number for each group and the p values (t-test with Welch’s correction) are shown within the graph. Data are presented as mean ± SEM. b, c Echocardiographic measurements demonstrate that the LV ejection fraction (EF) and fractional shortening (FS) are restored to normal values in the mdx:utr −/− :sln +/− and tKO mice. ** p < 0.0001 vs. other groups; n = 6 per group. Data are presented as mean ± SEM (t-test with Welch’s correction)

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  Fig. 6. Postnatal AAV9.shSLN treatment ameliorates DMD and associated cardiomyopathy in mdx:utr −/− mice. 1-month old male and female mice are injected with AAV or saline and experiments are performed 12 weeks post-injection. a, b Representative western blots show that AAV9.shSLN treatment effectively reduces SLN expression in skeletal (pectoral muscle) and cardiac (ventricle) muscles. Uncropped scans of western blots are shown in Supplementary Fig. 8d, e. The AAV treatment reduced SERCA2a and CSQ levels in the pectoral muscles of mdx:utr −/− mice. c, d Quantitation of areas with mononuclear infiltration in the H&E stained tissue sections show that cell necrosis is significantly reduced in both quadriceps and ventricles of AAV treated mdx:utr −/− mice compared to that of saline injected mdx:utr −/− controls. Data are presented as mean ± SEM. The n number for each group and the p values (t-test with Welch’s correction) are shown within the graph. e LV EF, f forelimb muscle strength, and g, h the maximum twitch force generated by EDL and hemidiaphragm at 2 Hz are significantly improved in the AAV treated mice compared to that of saline injected mdx:utr −/− controls. **p < 0.0001 vs. other…