Generation of Vma21 conditional knockout mice strains

Vma21 conditional knockout mice were generated using the CRISPR-Cas9 system to introduce 2 loxP sites, flanking upstream of promoter and last intron, into the same allele of the X-linked Vma21 gene. We designed gRNAs targeting upstream from the Vma21 promoter and its last intron and oligonucleotides, containing loxP sites and BamHI or HindIII restriction sites, flanked by 60 bp homologous to the targeted regions. Zygotes were microinjected with a mixture of Cas9 mRNA, gRNAs, and oligos and transferred to pseudopregnant female mice. Genotyping of 8 F0 born pups using tail-extracted DNA and Vma21-specific primer sets flanking the loxP sites led to the identification of one female founder mouse carrying the loxP sites on both alleles of the X chromosomes. The founder mice were healthy, normal in size, and did not display any phenotype compared to their wild-type littermates.

Animals and experimental protocols

Control [(C57BL/6, Stock No: 000664), ACTA1p-cre/Esr1 (Tg(ACTA1-cre /Esr1*)2Kesr/J, Stock No: 025750 HSA-MCM), and B6.FVB(129S4)-Tg(Ckmm-cre)5Khn/J, Stock No: 006475] mice were purchased from Jackson Laboratories. All animal experimental protocols were approved by the Animal Studies Committee of Washington University School of Medicine per IACUC guidelines. Mice were housed in a temperature-controlled environment with 12-h light–dark cycles where they received food and water ad libitum. Tamoxifen-supplemented water was prepared by dissolving 1 g tamoxifen citrate (Goldbio; T-750–2) into 2L of DI water.

Genotyping of mice

Genomic DNA was extracted from mutants and control mice tails using KAPA Express DNA extraction Kit (Kapabiosystems, KK7103) and standard PCR was performed using Lambda Biotech (206,811) and gene-specific primers. PCR products were subjected to electrophoresis on a 2.5% agarose gel. After traditional PCR verification of germline transmission of X-linked floxed alleles in F1 litters, mouse genotypes were determined by real-time PCR analysis (Transnetyx, Cordova, TN, USA) using tail biopsy samples.

Histochemistry/immunofluorescence

For histological analysis of hearts, mice were transcardially perfused with phosphate-buffered saline (PBS; Gibco, 14,190–136). Hearts were dissected and placed individually into scintillation vials containing sufficient 3.7% paraformaldehyde (PFA) to fully submerge the tissue, and immersion-fixed overnight at 4 °C. Following fixation, hearts were rinsed three times in PBS and cryoprotected by immersion in 30% (w/v) sucrose in PBS overnight at 4 °C. Tissues were then embedded in optimal cutting temperature (OCT) compound, frozen on dry ice, and stored at − 80 °C until ready to be sectioned to a 10 μm thickness.

For histological analysis of skeletal muscle, samples were mounted in tragacanth gum (10% solution, Sigma-Aldrich, G1128), flash-frozen in 2-methylbutane over liquid nitrogen, and stored at − 80 °C until ready to be sectioned to an 8 μm thickness.

For the H&E stain, a 1% aqueous solution of eosin Y (Sigma E-6003) was prepared in deionized water, and Harris hematoxylin stain (Lerner Laboratories, 1,931,382) was filtered before use. Slides in a metal staining rack were immersed in the filtered Harris hematoxylin for 10 s, then transferred to a beaker of tap water, and rinsed until the water was clear. Then, the slides were immersed in eosin stain for 30 s and again rinsed with tap water. Then, sections were dehydrated in ascending alcohol solutions (50%, 70%, 80%, 95% × 2, 100% × 2) and cleared with xylene three or four times, and a glass coverslip was mounted to the glass slide using Permount.

For esterase enzyme histochemistry, cryosections of snap-frozen skeletal muscle (8 μm) were incubated at room temperature in a staining solution containing α-naphthyl acetate and pararosaniline-based coupling reagents prepared fresh prior to use, as previously described. Sections were then rinsed in water, dehydrated through graded alcohols, cleared in xylene, and mounted with Permount.

For acid phosphatase enzyme histochemistry, Sects. (8 μm) were incubated in a naphthol phosphate-based substrate solution containing diazonium coupling reagents prepared fresh prior to use, as described in standard protocols. Following incubation, sections were washed, dehydrated through graded alcohols, cleared in xylene, and mounted with Permount.

For immunostaining, sections were fixed using 3.7% PFA for 10 min followed by 10 min of ice-cold acetone. The muscle sections were then permeabilized for 10 min in 0.5% Triton X-100 and blocked for 1 h at room temperature in PerkinElmer blocking reagent (FP1012). Primary antibodies were diluted in blocking reagent and incubated at 4 °C overnight. After three rinses for 5 min each with 1 × PBS, secondary antibodies were added to the slides at 1:500 dilution in blocking reagent and incubated for 1 h at room temperature. Slides were rinsed with 1 × PBS again three times for 5 min each and then incubated for 10 min with 4′,6-diamidino-2-phenylindole (DAPI; 1 μg/ml) followed by a final three PBS rinses. A cover glass was mounted to slides using Mowiol 4–88 (Sigma-Aldrich, 81,381). The following antibodies and dilutions were used for immunostaining: rabbit anti-laminin (abcam; ab11575; 1:500), rabbit anti-Caveolin-3 (Thermo Fisher Scientific; PA1-066; 1:250); mouse anti-dystrophin (Millipore Sigma; D8043; 1:250), rat anti-LAMP2 (abcam; ab13524; 1:200), rabbit anti-LC3B (Millipore Sigma; L7543; 1:200), rabbit anti-SQSTM1 (Proteintech; 18,420–1-AP; 1:1000), mouse anti-Ubiquitin (P4D1) (Cell Signaling; 3936; 1:200), and mouse anti-C5b-9 (Dako; M0777; 1:200).

Frozen skeletal muscle Sects. (8 μm) from patients with genetically confirmed Vma21 mutations and from one control individual were analyzed. Sections were fixed in cold acetone (2 × 10 min) and incubated overnight at 4 °C with primary antibodies against C5b-9 (mouse monoclonal IgG2a, Dako M0777; 1:50) and CD63 (mouse monoclonal IgG1, Novus Biologicals NBP2-42,225; 1:250). After washing in phosphate-buffered saline (PBS), sections were incubated for 1 h at room temperature with isotype-specific secondary antibodies: Alexa Fluor 488 goat anti-mouse IgG2a (Invitrogen A21131; 1:250) and Alexa Fluor 568 goat anti-mouse IgG1 (Invitrogen A21124; 1:250). Nuclei were counterstained with DAPI, and sections were mounted in Mowiol.

Imaging and image analysis

Immunofluorescently labeled cardiac and skeletal muscle sections were imaged using a Nikon CSU-W1 spinning disk confocal microscope (Nikon Instruments, Melville, NY). Images were acquired using NIS-Elements HC software (Nikon Instruments). Human skeletal muscle sections were imaged using a Zeiss Axioplan 2 microscope equipped with an Axiocam 305 mono camera. Image acquisition settings were kept identical across samples within each experiment. Hematoxylin and eosin (H&E)-stained mouse heart and skeletal muscle sections were imaged using a Leica DM4 B microscope (Leica Microsystems, Germany), and images were acquired using Leica Application Suite X (LAS X) software.

For histological quantification, three non-overlapping fields per sample were acquired at 40 × objective (400 × total magnification). Percentage of fibers with internal nuclei, percentage of fibers exhibiting splitting, and fiber size variability (percent coefficient of variation) were quantified using Fiji (ImageJ, National Institutes of Health).

Transmission electron microscopy

Tibialis anterior, gastrocnemius, and quadriceps muscles were dissected from control and HSA-Vma21KO mice after 4 months of tamoxifen treatment and fixed overnight at 4 °C in fixative solution (2.5% glutaraldehyde, 2% PFA, 0.15 M cacodylate buffer, pH 7.4, with 2 mM CaCl2). After fixation, samples were rinsed in 0.15 M cacodylate buffer containing 2 mM calcium chloride three times for 10 min each followed by a secondary fixation in 1% osmium tetroxide and 1.5% potassium ferrocyanide in 0.15 M cacodylate buffer containing 2 mM calcium chloride for 1 h in the dark. The samples were then rinsed three times for 10 min each in ultrapure water and en bloc stained with 2% aqueous uranyl acetate overnight at 4 °C in the dark. After four washes for 10 min each in ultrapure water, the samples were dehydrated in a graded acetone series (10%, 30%, 50%, 70%, 90%, 100% × 3) for 10 min each step, infiltrated with Spurr’s resin (Electron Microscopy Sciences), and embedded and polymerized at 60 °C for 48 h. After curing, 70-nm-thin sections were cut and imaged on a TEM (JEOL JEM-1400Plus) at 120 kV.

Western blotting

Heart or muscle tissue lysates were collected in RIPA buffer with protease inhibitor cocktail. Protein concentrations were obtained through BCA assay and measured with the BioTek Epoch plate reader at 562 nm. Each lane of a SDS-PAGE gel was loaded with 20 to 30 μg of total protein, and gels were run at 80 V for 2 h and 20 min. Next, the samples were transferred to PVDF membranes at 0.1 mA per rig overnight. Membranes were blocked with 5% milk for 1 h, and then primary antibodies were added and incubated overnight at 4 °C. Blots were treated with horseradish peroxidase secondary antibodies at a 1:5000 dilution in 5% milk for 1 h at room temperature and then rinsed three times with 1 × PBS-Tween before imaging membranes. Membranes were imaged using enhanced chemiluminescence (Cytiva Amersham, RPN2209) and a Syngene G:Box Chemi XT4 imager. The following antibodies and dilutions were used for western blotting: rabbit anti-VMA21 (abcam; ab242115; 1:250), rat anti-LAMP2 (abcam; ab13524; 1:500), rabbit anti-LC3B (Millipore Sigma; L7543; 1:500), mouse anti-SQSTM1 (Novus Biologicals; H00008878-M01; 1:1000), mouse anti-Ubiquitin (P4D1) (Cell Signaling; 3936; 1:1000), and rabbit anti-GAPDH (Cell Signaling; 2118; 1:1000). The following gel percentages were used: 15% gels for LC3B and VMA21; 10% gels for SQSTM1, Ubiquitin (P4D1), and LAMP2.

Inverted screen (mesh) test

Muscle strength and motor coordination were assessed using the inverted screen test. The apparatus consisted of a wire mesh grid (12 mm × 12 mm squares made of 1 mm diameter wire) mounted within a PVC frame (52 cm × 21.5 cm). Mice were placed in the center of the mesh and allowed to grip the grid with all four limbs. The screen was then inverted 180° and held approximately 40–50 cm above a padded surface. The latency to fall was recorded, with a maximum cutoff time of 60 s. Each mouse was tested in two trials separated by a 2–5 min rest period, and the average latency to fall was used for analysis.

Transthoracic echocardiography

Non-invasive ultrasound examination of the cardiovascular system was performed using a Vevo Ultrasound System (VisualSonics Inc, Toronto, Ontario, Canada) according to the following procedures. First, mice were lightly anesthetized with an intraperitoneal injection of 2% Avertin (tribromoethanol, 0.005 ml/g). Hair was removed from the anterior chest with a combination of shaving and chemical hair remover, and the animals were placed on a warming pad in a left lateral decubitus position to maintain normothermia. Ultrasound coupling gel was applied to the chest. Cursory examination of cardiac structure and function under physiologic conditions was obtained with hand-held manipulation of the ultrasound transducer. Complete two-dimensional, M mode, and Doppler ultrasound examination was performed from multiple views.

Statistical analysis

All quantitative data were analyzed using GraphPad Prism (GraphPad Software). Data are presented as mean unless otherwise indicated. Comparisons between two groups were performed using an unpaired two-tailed Student’s t-test. Experiments involving two independent variables were analyzed using two-way analysis of variance (ANOVA) followed by Šidák’s multiple comparisons test. Survival analysis was performed using Kaplan–Meier survival curves, and statistical significance between groups was assessed using log-rank (Mantel–Cox) test. A p value < 0.05 was considered statistically significant. In all cases, *P < 0.05, **P < 0.01, and ***P < 0.001.