DNM2 levels normalization improves muscle phenotypes of a novel mouse model for moderate centronuclear myopathy

Figures

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  Figure 1. Creation and validation of Dnm2R369W/+ mice (A) Dnm2 exonic representation and the respective DNM2 protein domains: GTPase, middle, pleckstrin homology, GTPase effector, and proline rich. The exons 10a, 10b, 12b, and 13b can be alternatively spliced. DNM2 R369W mutation was knocked in by CRISPR-Cas9 in the exon 8. The shDnm2 that was used in this study to downregulate DNM2 targets exon 4. (B) Chromatopherograms of WT and Dnm2R369W/+ mice, which present a GAG>GAA synonym mutation for genotyping purpose and the Arg>Trp R369W CNM mutation (CGC to TGG). (C) Example of genotyping with amplicons of different molecular weights: the WT allele as a 427-bp band and the Dnm2R369W mutated alleles as two bands of 243 bp and a 184 bp, generated by the cleavage of the Glu GAA site by the restriction enzyme XmnI. (D) Proportion of genotypes in the mouse offspring at 8 weeks of life (n = 30 mice). Chi-square test, p = 0.75. (E) Body weight (n = 10 up to 8 weeks, n = 5 from 12 to 24 weeks). (F) Dnm2 mRNA expression of pan (ubiquitous + muscle-specific), muscle-specific (exon 12B), and ubiquitous and Dnm2 isoforms (n = 5). (G) Absolute quantification of mutated and WT forms of Dnm2 by ddPCR.…

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  Figure 2. Muscle strength tests and deficits in the contractile properties (A) Four limbs grip test and (B) hanging test (n = 10 up to 8 weeks, n = 5 from 12 to 24 weeks). (C) Representative rotatory movements of the tail and forelimbs during the hanging test at 6 weeks of age (see Video S1). (D) TA muscle resistance to fatigue after sciatic nerve stimulations at 40 Hz. (E) Submaximal and maximum absolute force outputs and (F) maximum absolute force and (G) maximum specific force (n = 5 at 8 weeks old). The maximum force was calculated at 100 Hz. The specific force is expressed as the ratio of the force produced (mN) by the cross-sectional area of the muscle analyzed (CSA = muscle weight (mg)/muscle length (mm) x muscle density 1.06 mg/mm3). Mean ± SEM, Mann-Whitney or unpaired t test, ∗p < 0.05 and ∗∗p < 0.01: Dnm2R369W/+ differs from WT.

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  Figure 3. Muscle hypotrophy and histological hallmarks of centronuclear myopathy (A) Ratio of hindlimbs muscle weight per body weight at 8 and 24 weeks of age (8w and 24w). GA, gastrocnemius; TA, tibialis anterior; SOL, soleus; EDL, extensor digitorum longus. Analyses of muscle sections at 8 weeks old, with 2 mm2 analyzed per muscle. The TA muscles sections stained with hematoxylin and eosin were used for determining (B) the general histological aspect (n = 5), (C) the percentage of fibers presenting internalized nuclei (n = 3), and (D) the fiber diameter (n = 4). (E) SDH oxidative staining showed abnormal central accumulation (c), necklace fibers (n), or spokes of wheels aspect (s) (n = 3). Mean ± SEM, Mann-Whitney or unpaired t test, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, and ∗∗∗∗p < 0.0001: Dnm2R369W/+ differs from WT at the same age; α p < 0.05, αα p < 0.01, and ααα p < 0.001: Dnm2R369W/+ at 8 weeks differs from Dnm2R369W/+ at 24 weeks; n p < 0.05: Dnm2R369W/+ necklace fibers at 8 weeks differs from Dnm2R369W/+ 24 weeks. Bars represent 50 μm.

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  Figure 4. Muscle structure disorganization in Dnm2R369W/+ mice (A) Different populations of mitochondria and triads found in electron microscopy: normal mitochondria (NM), enlarged mitochondria (EM), damaged mitochondria (DM, with abnormal cristae), normal triad (NT) and altered T-tubule (AT) (n = 2). Bars in the larger panel and mitochondria represent 1 μm; bars in the triads represent 0.1 μm. (B) Prohibitin protein levels (∼30 kDa, n = 3), (C) mRNA level of Sirt1 and protein levels of (D) PARK2 (∼55 kDa, n = 4), and (E) phosphorylated and total S6K (60 kDa, n = 5). (F) BIN1 and DHPR localization in longitudinal muscle sections (n = 3, bar represents 10 μm) and (G) BIN1 protein expression (n = 5, ∼60 kDa). (H) Desmin mislocalization (arrows) in transverse muscle sections (n = 3). Mice at 8 weeks old. Bar represents 50 μm. Mean ± SEM. Unpaired t test, ∗p < 0.05 and ∗∗p < 0.01: Dnm2R369W/+ differs from WT.

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  Figure 5. Alterations in muscle fiber metabolism in Dnm2R369W/+ mice (A) TA muscle fibers positive for myosin isoforms IIa (MYH2) and IIb (MYH4) per muscle section (total area of 2 mm2). Negative or light labeling were considered mixed fiber type or type IIx/d. No fiber type I fiber (MYH7) was found. (B) qRT-PCR of genes coding for myosins type IIa, IIx/d, and IIb (MyH2, MyH1, and MyH4, respectively; n = 4) and (C) PYGM (n = 8, ∼100 kDa), (D) GAPDH (n = 8, ∼37 kDa), and (E) OXPHOS (n = 5, ∼15, 25, 35, 40, and 55 kDa) protein levels. Mice at 8 weeks old. Bar represents 100 μm. Mean ± SEM, Mann-Whitney test or unpaired t test: ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001: Dnm2R369W/+ differs from WT.

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  Figure 6. Intramuscular injections of AAV-shDnm2 normalized DNM2 expression, rescued the histopathology, and improved muscle force and ultrastructure Injections of TA muscles at 4 weeks old, muscles analyzed at 8 weeks old. (A) Dnm2 mRNA and (B) DNM2 protein expression (n = 5–6). (C) TA muscle weight (n = 6). (D) Transversal muscle sections stained with hematoxylin and eosin and SDH (c: central accumulation) (n = 4). (E) Sections stained with H&E were used to calculate fiber diameter (n = 4). (F) Quantification of SDH staining (n = 3). (G) Electron microscopy of TA muscles from Dnm2R369W/+ mice injected with AAV-shDnm2 presenting a mix of enlarged mitochondria (EM) and normal triads (NT) (n = 2). (H) Maximum absolute force and (I) specific force at 100 Hz (n = 6). Mean ± SEM. DNM2 protein level: Wilcoxon matched pairs test, ∗p < 0.05: Dnm2R369W/+ shDnm2 differs from Dnm2R369W/+ scramble; Mann-Whitney test, α p < 0.05: Dnm2R369W/+ scramble differs from WT scramble. Central staining ∗p < 0.05: Dnm2R369W/+ shDnm2 vs. Dnm2R369W/+ scramble; αααα p < 0.0001: Dnm2R369W/+ shDnm2 vs. WT shDnm2 or Dnm2R369W/+ scramble vs. WT scramble. Others: unpaired t test, α p < 0.05, αα p < 0.01, and ααα p <…

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  Figure 7. Improvement in CNM-like muscle phenotypes of Dnm2R369W/+ mice after DNM2 normalization with shDnm2 (A) Summary of the mean results obtained in WT and Dnm2R369W/+ mice at 8 weeks old submitted to a single injection of AAV-shDnm2 in the TA muscle and scramble in the contralateral TA at 4 weeks old. 100% represents the normalized values for WT muscles injected with scramble. (B) Mean results obtained for the characterization of Dnm2R369W/+ mice compared to previously published Dnm2-mutated CNM mouse models at 8 weeks old: the severe Dnm2S619L/+30 and the mild Dnm2R465W/+.28 The data are normalized to the WT values originally reported for each model. ∗No hanging was test described in the original publication of Dnm2R465W/+ mice, but it was assumed as equal to the WT. Some of these parameters were not analyzed in (A) as the treatment was intramuscular (for body weight and hanging test) or as centralization of nuclei appeared later than the age of analysis.