CRISPR-Cas-9 gene editing, PCR and genotyping
Using the CRISPR design tool at http://crispr.mit.edu we designed CRISPR RNA guides targeting the exon 40a locus of murine dysferlin. PAM sequence is shown in Additional file 1: Fig. S1A. To avoid issues with off-target effects, we chose to use the paired guide/Cas-9 nickase strategy when creating our mouse model. Guide RNA and Cas-9n mRNA were co-injected into pronuclear C57BL/6 embryos and the resultant pups were screened by PCR across the exon 40a locus. PCR primers (Sigma) used for genotyping are shown in Additional file 1: Fig. S1A and Additional file 8: Table 1. The 1 × PCR mix constituted 15µ L of 2 × Buffer D (Astral Scientific), 12 µL milli-Q water, 1 µL forward primer, 1 µL reverse primer, 0.2 µL Taq DNA polymerase (Invitrogen) and 1 µL of template gDNA (~ 500 ng). PCR cycling conditions were as follows: (i) 95 °C–3 min, (ii) 30x (95 °C –30 s, 58 °C –30 s, 72 °C -1 min), (iii) 72 °C -5 min. PCR products were mixed with 5 × DNA loading buffer (Bioline Meridian Bioscience) and run alongside a 100 bp ladder (Bioline Meridian Bioscience) on 1.8–3% TAE gel at 100 V for ~ 1 h. Founder mice carrying identifiable gene-editing events in and around exon 40a were consequently bred to homozygosity so RNA and protein could be studied without the confounding effect of the normal/WT suite of dysferlin transcripts and isoforms. We generated three founder mice that each carried a different deletion in and around exon 40a (Additional file 1: Fig. S1) and derived a distinct mouse line from each founder. For further details on generation and characterisation of exon 40a knockout mice, refer to Additional file 7.
SDS PAGE and Western blotting
Western blotting was done as previously described [26, 39]. Briefly, muscle sections (10 mm2, 8 µm thick) were homogenised on ice in SDS lysis buffer (2% SDS, 125 mM Tris pH 7.5) supplemented with protease inhibitors (Sigma Aldrich). Twenty micrograms of protein was mixed (1:1) with 5X SDS loading buffer, heated at 95 °C for five minutes and then resolved on a 3–8% Tris–acetate polyacrylamide gel (Thermo Fisher Scientific Melbourne, VIC, Australia) alongside the Page Ruler™ Plus Protein Standard (Thermo Fisher Scientific). Samples were then transferred onto a polyvinylidene fluoride (PVDF) membrane (Merck Millipore, Bayswater, WA, Australia). After blocking the membrane in 5% skim milk/PBS/0.1% Tween-20 for 1 h, anti-dysferlin primary antibody (1 in 500, NCL Hamlet-CE, Leica Biosystems,Buffalo Grove, IL) or anti-GAPDH loading control (1 in 2000, Millipore Bioscience Research Reagents) was incubated overnight at 4 °C with shaking. The membrane was washed in PBS/0.1% Tween-20 prior to incubation in a species-specific horseradish peroxidase conjugated secondary antibody for 2 h at room temperature (R.T). The membrane was washed again, and the signal developed on an X-ray film following addition of a chemiluminescence substrate (GE Healthcare, Parramatta, Australia).
Immunoprecipitation of dysferlin and calpain cleavage
Immunoprecipitation was conducted as previously described by Redpath et al.  with minor modifications. Briefly, quadriceps muscle sections from WT, 40aKO lines 1-to-3 and BLAJ mice were lysed in RIPA buffer without calcium and supplemented with protease inhibitors. Endogenous dysferlin was immunoprecipitated with N-terminal rabbit polyclonal antibody Romeo (Epitomics Inc.) and protein G–sepharose beads (GE Healthcare) overnight at 4 °C. Dysferlin-bound sepharose beads were then either incubated with 20 active unit (A.U.) of purified recombinant calpain-2 at 30 °C for 3 min or left undigested, in the presence of 2 mM CaCl2. Digestion was quenched by reconstitution into SDS lysis buffer (2% SDS, 10% glycerol, 50 mM Tris, pH 7.4, and 10 mM dithiothreitol (Sigma-Aldrich). Samples heated to 94 °C for 3 min and dysferlin was detected by Western blot analysis with the C-terminal antibody Hamlet-1.
For histopathology analysis, gluteus, psoas, quadriceps and spinalis muscles were dissected from ~ 12 months old BLAJ, Dysferlin exon 40aKO and C57BL-6 WT mice. Muscles were frozen for 30 s in 2-methylbutane (Sigma Aldrich, Australia) cooled in liquid nitrogen. Samples were then stored at − 80 °C for future use. Transverse muscle sections (8 µm) were collected onto Superfrost Plus glass slides (Menzel-Glaser, Braunschweig, Germany) using the cryostat CM1950 (Leica Biosystems).
Haematoxylin and Eosin (H&E) and Oil Red O Staining
H&E and Oil-Red O histochemical staining on frozen muscle sections was performed as previously described by Sullivan et al. . Images were acquired using the Aperio ScanScope CS (Leica Biosystems) at × 20 magnification and visualised using the Aperio Image Scope companion software (version 18.104.22.1680, Lecia Biosystems). The Aperio Image Scope ‘Colour Deconvolution v9’ algorithm was used to analyse the percentage of red stained (Oil-Red O positive) areas in each muscle section. However, it must be noted that Oil-Red O staining on cryosections is confounded by smearing of lipids across the section, which makes it difficult to quantify and to determine whether lipid droplets were localised in myofibers or adipocytes . To help overcome this caveat, the staining intensity and area of Oil-Red O-stained areas was accounted for to calculate the staining index.
Wheat-Germ Agglutinin (WGA)/DAPI Staining
To assess the presence of centralised nuclei in muscle sections, the sarcolemma was stained with WGA Alexa Fluor™ (AF)-488 conjugate (Thermo Fisher Scientific, Australia) and DAPI (1 µg/ml PBS, Thermo Fisher Scientific, Australia) was used to stain nuclei. Tissue sections were fixed in ice-cold 4% paraformaldehyde (PFA) for 10 min, washed twice for three minutes in PBS and then blocked in 2% BSA/PBS for 30 min prior to incubation in WGA-AF488 (diluted 1 in 200 Hank’s Buffered Saline Solution) for 10 min at R.T. Slides were washed three times for five minutes in PBS, counterstained with DAPI for five minutes then washed again in PBS before mounting and coverslipping in FluorSave™ Reagent. Non-overlapping images covering the entire muscle section were captured at × 10 magnification using the Leica DMi8 microscope (Leica Microsystems). A custom written Image J macro (Additional file 7) was used to enumerate centralised nuclei. Briefly, for every fiber, the macro detected its outline, measured the size of the fiber (area and minimum Feret diameter), then shrunk it slightly to exclude peripheral nuclei before counting the number of central nuclei.
Collagen VI and Dysferlin immunofluorescent staining
Following fixation in ice-cold 4% PFA for 10 min, slides were washed in PBS, fixed in 1:1 methanol/acetone for 10 min then blocked in 2% BSA/PBS for 30 min. For dysferlin staining, prior to fixation, muscle sections were subjected to heat mediated antigen retrieval as previously described by Roche et al. . In addition, slides were blocked with goat-anti-mouse Fab fragments (1 in 20, Jackson ImmunoResearch Laboratories) for 2 h at R.T or overnight at 4 °C, prior to the addition of primary antibody. Slides were incubated in primary antibody for 2 h at R.T or overnight at 4 °C. Collagen VI was detected using 1 in 2000 affinity purified rabbit polyclonal collagen type VI antibody (Fitzgerald Industries International), dysferlin was detected using NCL-Hamlet-1 (1 in 50). Following primary antibody incubation, slides were washed three times for five minutes in PBS, blocked in 2% BSA/PBS for 10 min then incubated in secondary antibodies goat anti-rabbit AF-488 or donkey anti-mouse AF-488 (1 in 200, Thermo Fisher Scientific) for 2 h at R.T. Slides were washed in PBS, counterstained with DAPI (1 µg/ml PBS) for five minutes (and WGA-AF555 when staining for dysferlin), washed again in PBS and finally mounted and coverslipped in FluorSave™ Reagent. Images were acquired on the DMi8 microscope under × 20 and × 40 objectives. The percentage of collagen VI positive stained areas was estimated using a custom written macro in Image J (Additional file 7).
Transmission electron microscopy imaging
Muscles previously frozen in 2-methylbutane were removed from the -80 freezer, cut into ~ 2mm2 transverse cross sections at -20 °C and immersed in cold 2.5% glutaraldehyde in 0.1 M cacodylate buffer overnight. Samples were washed in 0.1 M cacodylate buffer and post fixed with 2% osmium tetroxide for 2 h, dehydrated in a series of ethanol and embedded in TAAB Low Viscosity Resin (TAAB Laboratories). Sections were cut at 90 nm using a UC7 ultramicrotome (Leica Microsystems), mounted on formvar coated nickel grids, and stained with 2% uranyl acetate in 50% ethanol (10 min) and Reynold’s lead citrate (4 min). Grids were examined with a Jeol-1400 transmission electron microscope (JEOL Ltd., Tokyo, Japan) and micrographs recorded using a Jeol sCMOS Flash camera.
Lipidomics analysis was conducted on the quadriceps muscle frozen on dry ice after harvest from 18wk old 40aKO lines, WT and BLAJ mice. Approximately 20 mg of muscle tissue from the mid-belly of the quadriceps was sampled from each mouse and shipped on dry ice to Metabolomics Australia at The University of Melbourne. To prepare samples for lipid extraction and LC–MS/MS analysis, 20 mg of muscle tissue was placed in a cryomill tube and 600 µL of chilled methanol:chloroform (9:1) was added, including internal standards. Samples were homogenised using the cryomill set at 6800 rpm for 3 × 30 s cycles. Homogenates were transferred into new 2 mL Eppendorf tubes and 680 μL of 100% chloroform was added and thermomixed at 1000 rpm/20 °C for 15 min. Samples were centrifuged at 15,000 rpm at 0 °C for 10 min and then transferred to fresh 2 mL Eppendorf tubes before being placed in a speedvac to allow supernatants to evaporate to complete dryness. Finally samples were reconstituted in 100 µL of water-saturated butanol:methanol (9:1). Lipid analysis was done using the LCMS_C18_MRM (positive mode) method on the Agilent QQQ 6490 mass spectrometer. Agilent Mass Hunter QQQ Quant was used to quantify individual lipid species. Statistical data analysis and data normalisation was further carried out in MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/docs/Tutorials.xhtml). Data was normalised by median log transformation with mean centering and no filter applied prior to generating PCA curves and heatmaps from the.cvs file of raw intensity values.
Proteomic analysis was conducted on the contralateral quadriceps muscles of 18wk old 40aKO lines, WT and BLAJ mice used for lipidomics analysis. Muscles were frozen in 2-methylbutane after harvest. To prepare samples for proteomic analysis, 10 muscle sections (10mm2, 8 µm thick) per animal were homogenised on ice in 200 µL of 2%SDS/50 mM TEAB lysis buffer (pH 7.5) with protease inhibitors. After adding lysis buffer, samples were vortexed and then sonicated three times, cooling the samples on ice after each round of sonication. Samples were then frozen on dry ice and sent to the Proteomics facility at The Children’s Medical Research Institute (CMRI). LC–MS/MS analysis of muscle tissue lysates was performed on Dionex UltiMate 3000 RSLC nano system and Q Exactive Plus hybrid quadrupole-orbitrap mass spectrometer (Thermo Fisher Scientific) as described in supplementary methods. The resulting proteomics raw data was subjected to the Remove Unwanted Variation (RUVIII) method  from the RUV R package  to minimise batch-batch experimental variation, as further explained in Supplementary material. The processed raw LC–MS/MS data was then further analysed using MetaboAnalyst 5.0 (https://www.metaboanalyst.ca/docs/Tutorials.xhtml). Data was normalised by mean centering with no filter applied before generating PCA curves and heatmaps from the.cvs file of log transformed values.
Membrane repair assay
A cohort of 40aKO mice were shipped to the Ohio State University in the United States, for membrane repair assays (M20_1074). FDB muscles were surgically isolated from 40aKO mice, including age (4–6 months old) and sex-matched dysferlin null and WT control mice, according to protocols previously published by Guschina et al. . Briefly, intact FDB muscles were dissected at the tendons from animals and attached to 35 mm glass-bottom imaging dishes (MatTek) with liquid bandage (New-Skin). Muscles were surrounded with Tyrode’s solution containing 2.0 mM Ca2 + and 2.5 μM FM4-64 amphiphilic fluorescent styryl pyridinium dye (Life Technologies). An Olympus FV1000 multi-photon laser scanning confocal system was used to irradiate the cell membrane and induce a focal injury. A circular regional of interest on the cell membrane was selected for irradiation at 20–30% laser power for 5 s. Full field images were captured every 3 s, continuing for 60 s. Fluorescence intensity at the injury site in these images was determined using ImageJ software (NIH). Cell membrane repair kinetics were measured by establishing the change in fluorescence intensity (ΔF/F0). These values were calculated using the following equation for each image: (injury area fluorescence-background area fluorescence)/background fluorescence. To preclude potential experimental bias, all experiments were performed in a blinded fashion.
Statistical data analysis was performed using Graph-Pad Prism 9 software. Differences between genotypes was evaluated by One-Way ANOVA followed by Tukey’s multiple comparison test. Pairwise analysis was done using unpaired student t-test. P-values less than 0.05 were considered significant. Graphed data is represented as mean ± standard error of the mean (SEM).