Model generation and genotyping

The Hmz-NebΔExon55 mouse was generated in collaboration with The Centre for Phenogenomics (TCP) using CRISPR-Cas9 to delete 380 bp of intron sequence from the exon 55 deletion site of the previous NebΔExon55 mouse model (Fig. 1c, S1). Genotyping was performed using two PCR reactions to amplify either the WT or humanized deletion allele.

Primers:

WT allele.

Forward: GCATTCTTGCTCTTTCTTGTATGG.

Reverse: GAAAGGAACTCTGTCCTCTGG.

Hmz-NebΔExon55 allele.

Forward: AAGCTAGGGTGTTTGAGTCTCTTC.

Reverse: GACTGGAGCAACACACATTGTAC.

RNA sequencing

For the previous NebΔExon55 model, whole hind limb muscle was harvested at PN 2, and for Hmz-NebΔExon55, gastroc tissue was harvested at ~ 2 months of age and placed on dry ice before being stored at -80 C for later RNA extraction. A Qiagen RNeasy Fibrous Tissue RNA extraction kit was used to isolate RNA from gastroc tissue. RNA samples were sent to The Centre for Applied Genomics (TCAG) where they were prepared and sequenced. RNA underwent poly(A) enrichment and samples were prepped with New England Biolabs Next Ultra II Directional RNA-Seq for sequencing. RNA preps were sequenced using the Illumina NovaSeq 6000 platform at a depth of 100 million reads per sample. RNAseq read mapping and figure production was performed with the help of Lauren Liang from the Sickkids Centre of Computational Medicine (CCM) core. Neb gene read data was mapped to the GRC-m39 mouse genome assembly and analyzed in Integrated Genome Viewer (IGV). Custom mouse genomes were created to include either the residual FRT + vector sequence from the previous NebΔExon55 model or the minimally humanized deletion site in the new Hmz-NebΔExon55 model to accurately map the pseudoexon sequence. Sashimi plots were generated in ggsashimi according to Breschi et al. (2018) [14]. Junctions reads with a frequency below 3 were discarded. Transcript per million (TPM) junction reads encompassing Neb exons 54 to 56 were used to compare the proportion of transcript with a pseudoexon to that without.

General phenotypingAnimal care and monitoring

All animal procedures were performed in compliance with the Animals for Research Act of Ontario and the Guidelines of the Canadian Council on Animal Care. All protocols and procedures were pre-approved by The Centre for Phenogenomics (TCP). Animals were housed in appropriately temperature and light cycle controlled specific pathogen free conditions, in cages containing food, unlimited access to water, bedding material, and a plastic handling tube.

Daily welfare assessments were performed according to TCP standard procedure to determine if humane endpoint was met. No unexpected outcomes were observed leading to humane endpoint.

Body weight

Mouse body weights were measured twice weekly until ~ 2-months of age whereafter they were measured weekly until they reached endpoint.

Open field

Mice were allowed to acclimatize to the testing room in their home cages for 30 min prior to beginning open field testing. After 30 min, animals were placed in 43.5 × 43.5 cm open field chambers centrally illuminated to 250 lx. Chambers contained 16 beam IR rays (X, Y, and Z axes) to monitor mouse movement. Mouse movement was monitored over 20 min using Med Associates activity monitoring software. Horizonal movement data was collected according to X and Y axis beam breaks and rearing was measured according to the number of Z axes beam breaks.

Cryosectioning

TA and Quad muscle were harvested, and flash frozen in isopentane cooled by liquid nitrogen. Frozen muscle was stored at -80 oC. 8 μm horizontal and longitudinal sections were cut from the centre of frozen tissues at -30 oC on a Leica CM 1860 cryostat and affixed to Fisherbrand Superfrost Plus microscope slides. Slides were stored at -80 oC for staining.

Modified Gomori trichrome

Staining was performed according to https://www.newcomersupply.com/product/trichrome-stain-solution-gomori-one-step-light-green. In brief, 8 μm quadricep sections affixed to Fisherbrand Superfrost Plus slides were dried for 10 min, stained with 0.5% vector hematoxylin counterstain for 10 min then rinsed in tap water for 3 min. Slides were stained in gomori trichrome one step light-green (newcomer supply) at 39oC for 20 min. Slides were then rinsed with distilled water and differentiated in 0.25% acetic acid. Slides were dehydrated for 5 min in 95% ethanol followed by 5 min in 100% ethanol. Slides were cleared in xylene for 2 min then mounted with toluene before adding a glass cover slip and left dry overnight. Slides were imaged on Olympus BX43 light microscope at 40X magnification.

ImmunofluorescenceDystrophin fiber size IF and Nebulin and α-actinin double IF

Slides with muscle sections were brought to room temperature and dried for 2 min. tissues were fixed with cold 4% PFA at room temperature for 20 min. Slides were then washed 3 times in wash buffer (1x TBS, 0.1% Triton X-100, 0.1% Tween-20) in a staining jar on a tilter table for 5 min. Tissue was blocked in blocking buffer (1x wash buffer, 1% bovine serum albumin, 10% goat serum) for 1 h at room temperature in a moisture chamber. Slides were then incubated with 1/100 dilutions of primary antibody (Myomedix Neb N-term #6969, Abcam α-actinin A7811, Abcam dystrophin Ab15277) overnight at 4oC. The following day, slides were washed again following the previous wash step and then incubated with 1/1000 dilution of secondary antibody (Alexa fluor 488 (green) or 555 (red)) in blocking buffer at room temperature for 1 h in the dark. Slides were washed again following previous wash steps in the dark. ProLong Gold Antifade with DAPI mountant was added to the tissue and then sealed with a coverslip and left to dry in the dark for 24 h at room temperature before imaging.

N = 5–7 20X images per sample were taken from dystrophin stained slides on an Olympus BX43 microscope. Images were processed and fiber area and Ferets diameter was determined in ImageJ. Fibers with an area less than 300 were discarded to remove fiber assignment artifacts and fibers were binned by minimum Ferets diameter in GraphPad. 200X images from neb N-term and α-actinin co-stained slides were imaged on a Nikon A1R confocal microscope at a depth yielding maximal nebulin staining intensity.

Fiber typing IF

Staining was performed according to Luca J. Delfinis et al., (2022) [15]. In brief, slides with muscle sections were brought to room temperature and dried for 2 min. After drying, slides were treated with blocking buffer (wash buffer, 5% goat serum) for 1 h at room temperature. Slides were then incubated with 1/25 dilutions of primary antibody (DSHB MHCI BA-F8, MHCIIa SC-71, MHCIIb BF-F3) in blocking buffer overnight at room temperature. The following day, slides were washed in wash buffer for 20 min at room temperature on a tilter table. Next the slides were incubated with 1/1000 dilution of secondary antibody (alexa fluor 350 IgG 2b (blue), 488 IgGI (green), 568 IgM (red) in blocking buffer for 1 h in the dark. After secondary staining slides were washed in wash buffer for 20 min at room temperature in the dark. ProLong Gold Antifade mountant was added to the tissue and then sealed with a coverslip and left to dry in the dark for 24 h at room temperature before imaging.

N = 5–7 20X images were taken per sample from multi-MHC stained slides on an Olympus BX43 microscope. Fiber content was calculated manually in imageJ and pie charts and bar charts were generated in GraphPad.

Transmission electron microscopy

Thin longitudinal TA slices were taken from freshly harvested TA muscle and submersed in fixative containing 2.5% glutaraldehyde and 0.1 M sodium cacodylate buffer. Tissue was kept at room temperature for 20 min and then placed at 4oC overnight. The following morning samples were brought to the Advanced Bioimaging Center (The Hospital for Sick Childen). Here 90 nm thick sections were prepared on an RMC MT6000 ultramicrotome and then stained with uranyl acetate and lead citrate. Sections were then imaged on a FEI Tecnai 20 TEM microscope at 6000X and 30000X magnifications.

Protein analysisWhole protein analysis gel

Following Methods were modified from Kiss B et al., (2020) [8]. Flash-frozen tissues were pulverized in liquid nitrogen and then solubilized in urea buffer [8 M urea, 2 M thiourea, 50 mM tris-HCl, 75 mM dithiothreitol with 3% SDS, and 0.03% bromophenol blue (pH 6.8)] and 50% glycerol with protease inhibitors (0.04 mM E64, 0.16 mM leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride) at 60 °C for 10 min (Hidalgo, et al., 2009) [16]. Solubilized samples were centrifuged at 13,000 RPM for 5 min, aliquoted, flash-frozen in liquid nitrogen, and stored at − 80 °C. Nebulin expression analysis was performed on solubilized samples using a vertical SDS-agarose gel system (Hoefer SE600). 1% gels were run at 15 mA per gel for 3 h, then stained using Coomassie brilliant blue, and scanned using a commercial scanner. The scanned gels were subsequently analyzed with One-D scan (Scanalytics) and the optical density (OD) of Titin, Nebulin, and myosin heavy chain (MHC) was determined as a function of loading volume (in a range of six volumes). The slope of the linear relationship between OD and loading was obtained for each protein to quantify expression ratios. Nebulin and Titin expression levels were normalized to the MHC content, with final results normalized to the mean value of the MHC WT samples (Gineste, et al., 2020) [17].

Nebulin Western blotting

Solubilized samples were run on 0.8% SDS-Agarose gels and transferred onto polyvinylidene difluoride membranes using a semi-dry transfer unit (Trans-Blot Cell, Bio-Rad). Blots were stained with Ponceau S to visualize the total protein transferred. Blocking, detection with infrared fluorophore-conjugated secondary antibodies, and scanning followed recommendations for Odyssey Infrared Imaging System (LI-COR Biosciences). The following primary antibodies were used for Western Blotting: anti-nebulin N-terminal (1:1000; rabbit polyclonal; no. 6969, Myomedix). Protein expression was normalized to the MHC Ponseau S signal.

Intact muscle mechanics

Methods adapted from Li F et al., (2015) and Brynnel A et al., (2018) [18, 19]. Intact muscle mechanics were performed using the Aurora 1200 An ex vivo test system that has been described previously (Labeit, et al., 2010 and Ottenheijm, et al., 2009) [20, 21]. Briefly, muscles were attached between a combination servomotor-force transducer and fixed hook via silk suture in a bath containing oxygenated (95%/5% O2/CO2) Ringer solution (137 mM NaCl, 5.0 mM KCl, 1.0 mM NaH2PO4*H20, 1.0 mM MgSO4 * 7H20, 2.0 mM CaCl2 * 2H20, 24.0 mM NaHCO3, 11.0 mM glucose, pH 7.4, 30 °C. Optimal current was determined using twitches (pulse duration of 200 µs with biphasic polarity), under light tension and set 50% beyond what is required to induce a maximum twitch force. The optimal length (L0) was determined by adjusting muscle length until a maximal twitch force was produced. Active force was determined from a force–frequency protocol. The Sol muscle was stimulated at incremental stimulation frequencies 1, 5, 10, 20, 40, 60, 80, 100 and 150 Hz waiting 30, 60, 60, 90, 120, 120, 120, 120 and 120 s, respectively, in between each stimulation. The EDL protocol matched that of the Soleus, except for an additional force measurement at frequencies of 200 and 250 Hz. Muscle fatiguability was also measured by stimulating the soleus with a 40 Hz tetanus every 3 s for 74 repetitions. EDL fatigue was measured the same way using 60 Hz tetani. Measured force in mN were normalized by the physiological cross-sectional area (PCSA) of the muscle. The PCSA of the EDL and Soleus muscles were determined by using the measured muscle mass, muscle length, and taking the pennation angle of the fibers and the fiber length to muscle length ratio into account (Lieber and Ward, 2011) [22]. The PCSA was calculated as:

$$}\left( }}^}}} \right)}} } \left( } \right) } }\left( \theta \right)} \over } }}^}}}} } \left( }} \right)} \right)}}$$

(? is the pennation angle and ? is the physiological density of muscle).

From the force-frequency data, the maximal force produced, the minimal force produced, the time it takes to reach maximal force, the time the muscle takes to relax, and the frequency required to reach ½ of the maximal force can be extrapolated by fitting the force-frequency curve. The force-frequency curve was fit using the sigmoidal equation:

$$}_}}}\left( } \right)}}_}}}}\left( }_}}}}}_}}}} \over }\left[ }_}}}}} \over }}} \right]} \right\}}}} \right)$$

obtained from Prosser et al., 2011 where P0min gives the minimum specific force, P0max gives the maximum specific force, Fhalf defines the frequency where P0 = 0.5 of P0max, and 1/k is a measure of the steepness of the P0 vs. F relationship [23]. The curves for the different genotypes were also tested for significance using an extra sum of squares F-test. For fatigue, an index was used, where the average of the last 5 values measured were divided by the average of the first 5 values.

Thin filament length measurements

Muscles were rapidly excised and placed in relaxing solution (in mm: 20 BES, 10 EGTA, 6.56 MgCl2, 5.88 NaATP, 1 DTT, 46.35 K-propionate, 15 creatine phosphate, pH 7.0 at 20 °C) with 1% (w/v) Tritron X-100 and protease inhibitors for overnight on a 2D rocker at 4 °C. The solution was then replaced with fresh relaxing solution (without Triton) followed by 5 h in 50% glycerol/relaxing solution before storing at − 20 °C. Skinned muscles were placed in a sylgard dish containing 50% glycerol solution and dissected into fiber bundles. The ends of the bundles were attached to aluminum T-clips and the solution replaced with fresh relaxing solution. Bundles were stretched ∼30% of their base length. Relaxing solution was then replaced with 4% formaldehyde solution and muscles were fixed for overnight. After fixation, muscles were washed with phosphate buffer saline (PBS) and embedded in Tissue-Tek O.C.T.compound (Ted Pella Inc) and stored at − 80 °C. The O.C.T. embedded specimen was sectioned into 5 μm thick (Microm HM 550; Thermo Scientific) and placed on Super Frost Plus microscope slides. Fixed tissues were permeabilized again with 0.2% Triton X-100 in PBS for 20 min at room temperature on a light box to bleach out the background fluorescence. Washed with 1X PBS then incubated overnight at 4 C in dark humidity chamber with Alexa Fluor 488-conjugated Phalloidin (for actin staining 1:1000, A12379, Life Technologies) in PBS. The tissues were washed with PBS for 15 min at room temperature, followed by 2 rapid washes with ddH20. Coverslips were mounted onto slides with Aqua Poly/Mount (Polysciences Inc.). Images were captured using a Deltavision RT system (Applied Precision) with an inverted microscope (IX70; Olympus), a ×100 objective, and a charge-coupled device camera (CoolSNAP HQ; Photometrics) using SoftWoRx 3.5.1 software (Applied Precision). The images were then deconvolved using SoftWoRx. An average of 10 areas was observed for each tissue section. Thin filament lengths and sarcomere lengths were obtained from deconvolved images of EDL muscles stained with a fluorescently conjugated phalloidin antibody. Deconvolved images were reopened in ImageJ (http://rsb.info.nih.gov/ij), then the 1D plot profile was calculated. The plot profile was analyzed using Fityk 0.13.1(http://fityk.nieto.pl). A custom ‘rectangle + 2 half Gaussian’ function was used for analyzing phalloidin-stained images that consisted of a rectangle that was flanked by two half Gaussian curves. To account for actin overlapping in the Z-disk which creates a small bump in the center of the rectangle, the center points within the rectangle fit were de-activated. This improved the subsequent fit for the ‘rectangle + 2 half Gaussian’ function. Thin filament length was calculated as half the width of the rectangle plus half the width of the Gaussian fit at half maximum height. SL was calculated from the distance between the centers of two adjacent Gaussian fits. We analyzed a large number of images and determined thin filament length within the SL range of 2.4–2.8 μm. WT and HOM EDL fiber bundles from N = 3 male mice, 2 fiber bundles per animal.

Statistical analysis

Unless otherwise specified, the statistical analysis used includes either a two-tailed Student t-test (two-group single variable comparison) or one-way ANOVA (multiple-group single variable comparison) where relevant to determine the differences in group means.