Modeling muscle regeneration in RNA toxicity mice

Abstract

RNA toxicity underlies the pathogenesis of disorders such as myotonic dystrophy type 1 (DM1). Muscular dystrophy is a key element of the pathology of DM1. The means by which RNA toxicity causes muscular dystrophy in DM1 is unclear. Here, we have used the DM200 mouse model of RNA toxicity due to the expression of a mutant DMPK 3'UTR mRNA to model the effects of RNA toxicity on muscle regeneration. Using a BaCl2-induced damage model, we find that RNA toxicity leads to decreased expression of PAX7, and decreased numbers of satellite cells, the stem cells of adult skeletal muscle (also known as MuSCs). This is associated with a delay in regenerative response, a lack of muscle fiber maturation and an inability to maintain a normal number of satellite cells. Repeated muscle damage also elicited key aspects of muscular dystrophy, including fat droplet deposition and increased fibrosis, and the results represent one of the first times to model these classic markers of dystrophic changes in the skeletal muscles of a mouse model of RNA toxicity. Using a ligand-conjugated antisense (LICA) oligonucleotide ASO targeting DMPK sequences for the first time in a mouse model of RNA toxicity in DM1, we find that treatment with IONIS 877864, which targets the DMPK 3'UTR mRNA, is efficacious in correcting the defects in regenerative response and the reductions in satellite cell numbers caused by RNA toxicity. These results demonstrate the possibilities for therapeutic interventions to mitigate the muscular dystrophy associated with RNA toxicity in DM1.

Figure 1

RNA foci and RNA binding proteins expression in satellite cells. (A) PAX7 IF (green) and RNA FISH (red dots) in skeletal muscle from a DM1 patient show two PAX7+ve cells, one with an RNA foci. (B) PAX7 IF (green) with MBNL1 IF (red) shows an example of PAX7+ve cell with MBNL1 foci. (C) PAX IF (green) and RNA FISH (red dots) of the TA of DM200 D+ mouse showing PAX7+ve cells with RNA foci. PAX7 IF (green) and MBNL1 IF (red) of the TA from wild-type mouse (D) and DM200 D+ mouse (E) showing PAX7+ve cells express MBNL1. PAX7 IF (green) and MBNL2 IF (red) of the TA from wild-type mouse (F) and DM200 D+ mouse (G) showing PAX7+ve cells express MBNL2. DAPI stains nuclei (blue); arrows indicate PAX7+ve cells. Scale bar = 5 μm (for A–C) and 10 μm (for D–G).

Figure 2

Pax7 and myogenic differentiation markers are affected by expression of toxic RNA. qRT-PCR shows increased toxic RNA levels (GFP mRNA) in skeletal muscle (gluteus maximus) of DM200 mice induced with 0.2% doxycycline. Time course studies indicate increased GFP mRNA levels as early as 2 days post induction that increased over 10-fold as compared to uninduced mice (DM200 D−) by 1 week. Pax7 mRNA levels are significantly decreased in DM200 D− tissues and decrease further with induction of toxic RNA expression (DM200 D+). qRT-PCR shows changed expression of myogenic markers (Myf5, MyoG, Myod) in DM200 D− mice and with toxic RNA induction (DM200 D+) during the time course (0–14 days) of the experiment. n = 3 mice per group; ^*P < 0.05, ^**P < 0.01, ^***P < 0.001; Student’s t-test; error bars are mean ± SEM.

Figure 3

RNA toxicity reduced the number of satellite cells. (A) IF for PAX7 (red) in uninjured TA sections from DM200 D− and DM200 D+ mice. Nuclei were stained with DAPI (blue). Arrows represent PAX7+ve cells. Scale bar = 100 μm. (B) Quantification shows a decrease in PAX7+ cells per 100 fibers in skeletal muscle (TA) of DM200 D+ mice (2 weeks post-induction) as compared to DM200 D− mice. n = 3–4 mice per group; at least three non-overlapping sections/mouse; ^***P < 0.001; Student’s t-test; error bars are as mean ± SEM. (C) qRT-PCR shows decreased expression of Pax7 mRNA in skeletal muscle (TA) of DM200 D+ mice (2 weeks post induction) as compared to DM200 D− mice. n = 5 mice per group; ^*P < 0.05; Student’s t-test; error bars are mean ± SEM.

Figure 4

Skeletal muscle regeneration is delayed early by RNA toxicity. (A) Schematic showing the experimental design for single injury used in this study. (B) Representative images of H&E staining and PAX7 IF (green) at 5 days after injury by BaCl₂ in DM200 D− and DM200 D+ TA muscles. The H&E shows persistence of inflammatory response and lower number of regenerating fibers in DM200 D+ mice, and the PAX7 IF shows a deficit in satellite cell numbers in response to damage. (C) MYH3 IF(green) of TA muscles at 5 (left) and 7 (right) days post injury (dpi) demonstrates reduced and delayed expression of this marker of regeneration in DM200 D+ mice as compared to DM200 D− mice; laminin (red), nuclei are stained with DAPI (blue). (D) qRT-PCR shows that expression of GFP mRNA is induced to similar levels in control and BaCl₂ injected legs of DM200 D+ mice. At 5 days post BaCl₂, the levels of Pax7 and MyoD induction in response to damage are reduced significantly in the TA muscles of DM200 D+ mice as compared to DM200 D− mice. Saline represents the contralateral leg in each mouse, injected with PBS. n = 5 mice per group; ^*P < 0.05, ^**P < 0.01; Student’s t-test; error bars are mean ± SEM. Scale bars = 50 μm.

Figure 5

Muscle histology and fiber sizing confirm persistent delay in muscle regeneration and reduced satellite cell numbers in RNA toxicity mice. (A) Representative H&E staining of TA muscle sections 5, 14 and 28 days post injury (dpi) in the DM200 D− and DM200 D+ mice; scale bars = 50 μm. Graphs on right—the top graph represents quantification of the number of centrally nucleated fibers <30 μm (i.e. regenerating fibers) at 5 days post damage. The bottom two graphs at 14 and 28 days post damage represent a fiber size distribution plot with fibers <30 μm in the boxed area; n = 3–5 mice per group; five non-overlapping sections/mouse; ^*P < 0.05; Student’s t-test; error bars are mean ± SEM. (B) PAX7 IF (green) showing PAX7+ve cells (arrows) in DM200 D− and DM200 D+ TA muscles 28 days post injury; laminin (red); nuclei (blue); scale bars = 50 μm. Quantification shows a reduced (but not statistically significant) number of PAX7+ve cells in DM200 D+ mice; n = 3 mice/group; 5–6 non-overlapping sections counted/mouse; error bars are mean ± SEM.

Figure 6

Delayed regeneration persists to 56 days post damage. (A) Representative H&E staining of TA muscle sections 56 days post injury in the DM200 D− and DM200 D+ mice, showing more fiber size variability in the DM200 D+ mice; scale bars = 50 μm. Graph on right represents a fiber size distribution plot with fibers <30 μm in the boxed area; n = 3–5 mice per group; five non-overlapping sections/mouse; ^*P < 0.05; Student’s t-test; error bars are mean ± SEM. (B) PAX7 IF (red) showing PAX7+ve cells (arrows) in DM200 D− and DM200 D+ TA muscles 56 days post injury; laminin (green); nuclei (blue); scale bars = 50 μm. Quantification shows a significantly reduced number of PAX7+ve cells in DM200 D+ mice; n = 3 mice/group; 5–6 non-overlapping sections counted/mouse; ^***P < 0.001; Student’s t-test; error bars are mean ± SEM.

Figure 7

Repeated injury uncovers a dystrophic phenotype in skeletal muscles of RNA toxicity mice. (A) Schematic showing the experimental design used in this study. (B) H&E-stained sections of re-damaged TA muscles show increased fat droplets (white arrows) and a decreased number of regenerating fibers with persistent inflammatory response in DM200 D+ mice. Oil Red O staining confirms the increased presence of fat in TA muscles of DM200 D+ mice. MYH3 IF (an early fiber regeneration marker) (red) at 5 days post re-damage shows maturing fibers in wild-type mice (WT D+), while DM200 D+ mice show persistence of embryonic myosin expression. Laminin IF used for outlining muscle fibers (green), and nuclei stained with DAPI (blue). (C) IF for perilipin (a fat droplet surface marker) (red) shows increased adipogenesis in TA muscle sections from DM200 D+ mice as compared to WT D+ mice at 5 days post re-damage; nuclei are stained with DAPI (blue). (D) H&E staining and laminin IF (green) of TA muscle sections, with accompanying fiber size distribution quantification, show significantly smaller fibers in DM200 D+ mice as compared to WT D+ mice at 14 days post re-damage; n = 3 mice per group, ^**P < 0.01, Student’s t-test, error bars are mean ± SEM. (E) MYH2 IF (red) (a marker of fiber maturation) in TA muscle sections 14 days post re-damage (BaCl₂) shows decreased expression in DM200 D+ mice as compared to WT D+ mice. There was no difference in expression of MYH2 in non-injured muscle (saline) from the same DM200 D+ and WT D+ mice. Nuclei are stained with DAPI (blue). Scale bars = 50 μm.

Figure 8

LICA-ASO treatment improves muscle regeneration in RNA toxicity mice. (A) Schematic showing the experimental design used in this study. (B) H&E staining of TA muscle sections and (C) fiber size analysis show significantly increased size of central nucleated fibers (i.e. regenerating fibers) in hDMPK-ASO (IONIS 877864) treated mice as compared to control-ASO treated mice at 5 days after injury; n = 4 mice per group, ^**P < 0.01, Student’s t-test, error bars are mean ± SEM. H&E sections also show better resolution of inflammatory response and reduced fat droplets with hDMPK-ASO treatment. (D) IF for MYH3 (green) in TA muscle sections shows increased expression of MYH3 and larger size of regenerating fibers in hDMPK-ASO treated mice as compared to control-ASO treated mice at 5 days post injury; graph showing fiber size quantification of MYH3 positive fibers; n = 3 mice per group; ^*P < 0.05; Student’s t-test; error bars are as mean ± SEM. (E) IF for Pax7 (green) in TA muscle sections shows increased number of PAX7+ve cells (white arrows) in hDMPK-ASO treated mice as compared to control-ASO treated mice at d5 after injury. The graph shows number of PAX7+ve cells per 20× field; n = 4 mice per group; five non-overlapping fields/mouse; ^**P < 0.01; Student’s t-test; error bars are as mean ± SEM. Nuclei are stained with DAPI (blue). Scale bars = 50 μm.

Figure 9

Gene expression analyses with hDMPK-ASO treatment. qRT-PCR from TA muscles collected 5 days post BaCL₂ injury shows that the hDMPK-ASO reduced toxic RNA levels (GFP DMPK 3′UTR mRNA) in DM200 D+ mice as compared with mice treated with the control-ASO. qRT-PCR also shows increased expression of Pax7 (marker of satellite cells) and Myh3 (regeneration marker). There was no significant difference in the expression of Myod or Myog. qRT-PCR also shows significant decreased expression of Col1a1 mRNA (a marker of fibrosis). n = 4–5 mice per group; ^*P < 0.05, ^***P < 0.001; Student’s t-test; error bars are mean ± SEM.

Figure 10

LICA-ASO improves muscle regeneration and reduces dystrophic changes in RNA toxicity mice. (A) H&E staining of TA sections shows improved muscle histology in DM200 D+ mice treated with hDMPK-ASO as compared with control-ASO at 14 days post injury (dpi). Scale bar = 20 μm. (B) Gomori trichrome staining of representative TA muscle sections collected at 14 dpi shows decreased collagen staining (blue stain) in the hDMPK-ASO-treated DM200 D+ mice as compared to control-ASO treated mice. (C) Representative images of H&E staining and Gomori trichrome staining of TA muscle sections collected at 28 dpi from DM200 D+ mice treated with either control-ASO or hDMPK-ASO show decreased fibrosis and more intact muscle architecture in the hDMPK-ASO-treated mice. For B and C, scale bars = 50 μm.

Figure 11

LICA-ASO treatment rescues satellite cells in the RNA toxicity mouse. (A) qRT-PCR shows decreased expression of toxic RNA (GFP DMPK 3′UTR mRNA) and increased Pax7 mRNA in TA muscles of DM200 D+ mice treated with hDMPK-ASO as compared with control-ASO treated mice, at 28 dpi. n = 4–5 mice per group, ^*P < 0.05; Student’s t-test, error bars are mean ± SEM. (B) IF for PAX7 (red) and quantification of satellite cells (white arrows) in TA muscle sections collected at 28 dpi show that treatment with hDMPK-ASO rescued the defect in satellite cell number caused by RNA toxicity; compare WT D+ to DM200 D+ mice treated with control-ASO or hDMPK-ASO. Nuclei are stained blue with DAPI. n = 4–5 mice per group; 3–5 non-overlapping sections/mouse; ^***P < 0.001; Student’s t-test; error bars are as mean ± SEM. Scale bars = 50 μm.