Generation of a novel mouse model of nemaline myopathy due to recurrent NEB exon 55 deletion

Figures

• 

  Fig. 1. Pseudoexon identification and Hmz-NebΔExon55 model generation. (A) RNAseq Sashimi plots from n = 2 NebΔExon55 mice compared to a WT control spanning Neb exons 54–56. Numbers with connecting arches indicate splice junction reads, which are a measure of the number of transcripts from the indicated spliced products. With exon 55 deletion, it would be predicted that exon 54 is spliced directed to 56; however, in the NebΔExon55 mice, ~ 94% of the transcript includes intronic material between exons 54 and 56, with distinct splice donor and acceptor sites forming a pseudoexon. (B) Cartoon depiction of NebΔExon55 pseudoexon depicting the location of cryptic splice sites leading to the pseudoexon being spliced into the mature mRNA. Translated amino acid sequence of the pseudoexon contains two premature termination codons (PTC). The presence of these PTCs likely leads to transcript degradation through nonsense mediated decay (NMD), explaining the reduced Neb transcript levels in the NebΔExon55 mice. (C) Schematic of Hmz-NebΔExon55 model development whereby 380 bp of sequence encompassing the pseudoexon is replaced with a short 58 bp human intron 54 sequence containing a validated cas9 gRNA…

• 

  Fig. 2. Neb transcript restoration and protein expression. (A) RNAseq Sashimi plots from n = 4 Hmz-NebΔExon55 HOM animals encompassing Neb exons 54–56. Numbers with connecting arches indicate splice junction reads. There is a reduction to ~6.4% in pseudoexon inclusion in Hmz-NebΔExon55 mice, with 94% of transcripts favoring complete exon skipping of exon 55 with no additional intronic sequence. (B) RNAseq comparing Neb transcripts per million (TPM) reads between n = 2 WT and HOM NebΔExon55 mice and n = 4 WT and HOM Hmz-NebΔExon55 mice. Transcript reads indicate a decrease in HOM NebΔExon55 mice Neb transcript levels and no significant change in Hmz-NebΔExon55 mice Neb transcript levels. (C) Western blot with an antibody to the N-terminus of nebulin comparing Hmz-NebΔExon55 WT and Hmz-NebΔExon55 tibialis anterior (TA), extensor longus digitorum (EDL), and soleus (SOL) nebulin protein expression normalized to PonceauS (Pon(S)) myosin heavy chain (MHC) staining. n = 3 Hmz-NebΔExon55 mice see a reduction to 22.2% (TA), 23.9% (EDL), and 40% (SOL) WT nebulin protein expression levels.

• 

  Fig. 3. Hmz-NebΔExon55 survival and phenotyping. (A) Kaplan-Meier survival analysis comparing Hmz-NebΔExon55n = 10 pooled WT and HET mice to n = 9 Hmz mice demonstrating that survival is reduced in the Hmz animals with a median survival of 139 days. (B) Body weight over time comparing Hmz-NebΔExon55 sex and age matched pooled WT and HET mice to Hmz mice demonstrating a reduction in male and female Hmz mice body weights that is first noted at 3 weeks of age and that persists until at least 26.5 weeks of age (age of last measurement). (C) Photomicrograph comparing a ~ 3-month-old HET (left-brown) and Hmz-NebΔExon55 mouse (HMZ, right-black) depicting HOM animals having a visibly distinct smaller stature. (D) Mouse rearing at 30 and 60 days of age measured through open field testing. Comparison is between Hmz-NebΔExon55 (n = 6) mice and sex and age matched pooled WT and HET mice (30 day n = 12, 60 day n = 11). Hmz mice have a reduction of rearing at both 30 days of age and 60 days of age that is indicative of motor function deficits

• 

  Fig. 4. Hmz-NebΔExon55 intact muscle mechanics analysis. Intact muscle mechanics on male and female WT, HET, and HOM Hmz-NebΔExon55 EDL and SOL tissue. (A) Force frequency measurements on EDL and SOL muscle. Muscles were stretched to optimal length (L0) and then stimulated at incremental stimulation frequencies. Forces measure in mN and normalized to physiological cross-sectional area (PCSA) indicate that there is a large magnitude reduction in HOM EDL maximal force generation potential and a moderate reduction in HOM SOL maximal force generation potential that is more pronounced in male animals. (B) Muscle fatigue measurements on EDL and SOL muscle. Muscles were stretched to optimal length (L0) and then stimulated at constant 150 Hz (SOL) and 200 Hz (EDL) tetani every 3 s for 74 repetitions. Fatigue measurements indicate that there is a significant increase in fatigue resistance in EDL muscle (P = 0.0089) and a trend towards increased fatigue resistance in SOL muscle (P = 0.0832) compared to WT controls

• 

  Fig. 5. Hmz-NebΔExon55 TA fiber type composition shift. Comparing skeletal muscle fiber composition in (n = 4) ~ 2-month-old Hmz-NebΔExon55 male mice TA muscle. Isopentane flash frozen horizontal TA sections were stained for myosin heavy chain (MHC) type IIb, IIa, and I protein. n = 8 representative 20X magnification immunofluorescence images were taken per sample and quantified by hand in ImageJ for fiber content. HOM animals see a significant decrease (P = 0.029) in type IIb fast fiber composition and a significant increase in type IIa (P = 0.0429) and I (P < 0.001) slow fiber composition with no significant (P = 0.6114) change in type IIx composition

• 

  Fig. 6. Hmz-NebΔExon55 muscle tissue weight and fiber size reduction. (A) Muscle tissue weights for ~ 3-month-old Hmz-NebΔExon55 WT, Het, and HOM mice. HOM animals have a reduction in tibialis anterior (TA), extensor digitorum longus (EDL), plantaris (Plant), gastrocnemius (Gast), and quadriceps (Quad) muscle compared to WT and HET littermates indicative of hypotrophy. Conversely, HOM animals have either no change or else a slight increase in soleus (SOL) and diaphragm (Dia) muscle weights compared to WT and HET littermates. (B) Measuring skeletal muscle fiber size in (n = 4) ~ 2-month-old Hmz-NebΔExon55 male mice TA muscle. Isopentane flash frozen horizontal TA sections were stained for dystrophin, a membrane associated protein. n = 8 representative 20X magnification immunofluorescence images were taken per sample and quantified in ImageJ for minimum Feret’s diameter. Imaging indicates a significant shift to smaller fiber size composition within HOM animals compared to WT

• 

  Fig. 7. Hmz-NebΔExon55 nemaline rod formation and reduced thin filament length. (A) Modified gomori-trichrome stain on isopentane flash frozen Quad crossections from n = 2 ~ 2-month-old Het and HOM male Hmz-NebΔExon55 mice indicates that HOM mouse fiber size is reduced with apparent nemaline rod formation (yellow arrow). (B) TEM imaging of glutaraldehyde fixed longitudinal TA sections from n = 4 ~ 2-month-old WT and HOM Hmz-NebΔExon55 mice indicates clear nemaline rod formation throughout HOM tissue that is not present in WT. (C) Phalloidin staining ~ 3-month-old WT and HOM Hmz-NebΔExon55 thin filaments. At the bottom, a screen capture of a plot profile (yellow line) captured in ImageJ and how the plot profiles were fit with a rectangular gaussian in Fityk. (D) Thin filament length (TFL) measurements indicates that there is a shift to shorter TFL and sarcomeres in HOM animals. (E) WT/HOM t-test for TFL at sarcomere lengths of 2.4–2.8 μm indicates that there is a significant (P < 0.0001) TFL reduction from 1.06 μm in WT to 0.96 μm in HOM animals

• 

  Fig. 8. Nebulin distribution and localization. Double Immunofluorescence labelling for Neb N-terminus and α-actinin in n = 4 WT and HOM male Hmz-NebΔExon55 TA muscle. (A) Cross section indicates the proportion of higher nebulin expressing fibers is reduced in HOM mice. (B) Confocal imaging of longitudinal sections demonstrates that nebulin’s localization is normal in HOM mice. Note that nebulin expression in the Hmz muscle appears “thicker”, possibly as a result of the generally thickening of the Z band seen in these mice