A novel mouse model of Duchenne muscular dystrophy carrying a multi-exonic Dmd deletion exhibits progressive muscular dystrophy and early-onset cardiomyopathy

Figures

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  Fig. 1.. CRISPR/Cas9-mediated generation of Dmd Δ52-54 mice. (A) Exon map of Dmd Δ52-54. As a result of the mutation, exons 51 and 55 (orange) are out of frame. (B) sgRNAs compatible with the S. pyogenes Cas9 system were designed to target regions within Dmd intron 51 (in51g1 and in51g2) and intron 54 (in54g2 and in54g3). The founder Dmd Δ52-54 carried the deletion produced by in51g2 and in54g3 (green), leading to a deletion of 116 kb. (C) Genotyping with the corresponding primer sets (Table 1) detects deletion junctions present in Dmd Δ52-54 mice. The deletion junction was confirmed using Sanger sequencing, which also detected a one base pair insertion between Cas9 cut sites (highlighted in orange). WT, wild-type control; neg, PCR negative control; PAM, protospacer adjacent motif.

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  Fig. 2.. Dmd Δ52-54 mice lack dystrophin expression. (A) RT-PCR detection and Sanger sequencing of deletion junctions in Dmd transcripts in heart tissues of wild-type (WT) and Dmd Δ52-54 (Δ52-54) mice. mGAPDH is shown as a control. (B) Protein isolated from gastrocnemius and heart tissues showed absence of dystrophin in Dmd Δ52-54 mice. Anti-vinculin is used as the loading control. (C) Immunofluorescence staining detected the sarcolemma using laminin-α2 (green) and the presence of dystrophin (red) in wild-type and Dmd Δ52-54 cross sections of the indicated tissues. Scale bar: 100 µm. (D) Immunofluorescence staining probing for dystrophin, α-syntrophin and β-sarcoglycan in gastrocnemius cross sections of WT and Dmd Δ52-54 mice. Scale bar: 110 µm.

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  Fig. 3.. Dmd Δ52-54 displays dystrophic histology. (A) Hematoxylin and Eosin (H&E) staining of gastrocnemius cross sections from 12-week-old wild-type (WT) and Dmd Δ52-54 mice. Scale bar: 50 µm. (B) Quantification of centralized nuclei in 12-week-old wild-type (n=4) and Dmd Δ52-54 (n=5) mice. (C) Average minimum Feret's diameter and (D-F) distributions of minimum Feret's diameter were quantified using H&E cross sections from wild-type (n=4) and Dmd Δ52-54 (n=4-5) mouse lines. (G) Masson's trichrome staining was performed on gastrocnemius, triceps, diaphragm and heart tissues of 12-week-old wild-type (n=4-5) and Dmd Δ52-54 (n=4-5) mice, and the fibrotic area was quantified. Scale bar: 250 µm. All data are presented as the mean±s.d. Statistical analyses were performed using a Student's t-test. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

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  Fig. 4.. Dmd Δ52-54 mice exhibit dystrophic histology at 52 weeks. (A) Hematoxylin and Eosin (H&E) staining of gastrocnemius cross sections from 52-week-old wild-type (WT) and Dmd Δ52-54 (Δ52-54) mice. Scale bar: 50 µm. (B) Quantification of centralized nuclei in 52-week-old wild-type (n=4) and Dmd Δ52-54 (n=5) mice. Using H&E cross sections, (C) average minimum Feret's diameter and (D-F) distributions of minimum Feret's diameter were quantified from wild-type (n=3) and Dmd Δ52-54 (n=5) mouse lines. (G) Masson's trichrome staining was performed on gastrocnemius, triceps, diaphragm and heart tissues of 52-week-old wild-type (n=3-4) and Dmd Δ52-54 (n=5) mice, and the fibrotic area was quantified. Scale bar: 250 µm. All data are presented as the mean±s.d. Statistical analyses were performed using a Student's t-test. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

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  Fig. 5.. Dmd Δ52-54 mice exhibit elevated body weight and compromised muscle strength from 12 to 52 weeks. (A) Serum CK activity of Dmd Δ52-54 (Δ52-54) mice relative to wild-type (WT) mice at 12 and 52 weeks. (B) Body weight of wild-type (n=4) and Dmd Δ52-54 (n=13) mice at 12, 28 and 52 weeks of age. (C) Forelimb and hindlimb grip strength of wild-type (n=4) and Dmd Δ52-54 (n=13) mice at 12, 28 and 52 weeks of age. All data are presented as the mean±s.d. Statistical analyses were performed using Student's t-test. ns, not significant; *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001.

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  Fig. 6.. Dmd Δ52-54 mice develop long-term persistent cardiac hypertrophy and tachycardia. (A) Wheat germ agglutinin (WGA) staining of left ventricular cross sections was conducted in wild-type (WT) and Dmd Δ52-54 (Δ52-54) mice at 12 weeks (WT, n=5; Δ52-54, n=3) and 52 weeks (WT, n=4; Δ52-54, n=4). Scale bar: 50 μm. (B-D) The heart in wild-type and Dmd Δ52-54 mice was analyzed at 12, 28 and 52 weeks using echocardiography to determine left ventricular posterior wall thickness during systole (B), left ventricular systolic volume (C) and left ventricular end systolic diameter (D). (E) mRNA expression of Nppa in whole hearts of wild-type and Δ52-54 mice at 12 weeks. (F) Heart rate and (G) ejection fraction in wild-type and Dmd Δ52-54 mice was analyzed at 12, 28 and 52 weeks using echocardiography. In B-D and F-G, n=5-6 for WT, n=5-7 for 52-54. All data are presented as the mean±s.d. Statistical analyses were performed using Student's t-test. ns, not significant; *P<0.05; **P<0.01.