Animals
All animal procedures were conducted in accordance with guidelines for the care and use of laboratory animals as approved by the Institutional Animal Care and Use Committee (IACUC) of Children’s National Health System.
The mdx (C57BL/10ScSn-mdx/J) mouse model of DMD, utilized for all experiments, harbors a nonsense point mutation in exon 23 of the dystrophin gene and lacks dystrophin expression in muscle tissue. Four-week-old male mdx (n = 6) and wild-type (WT) C57BL/10 (n = 2) mice were purchased from the Jackson Laboratory (Bar Harbor, ME). All animals were housed at the Children’s National Health System (CNHS) Animal Facility in a vented cage system under 12-h light/dark cycles. Standard mouse chow and water were provided ad libitum.
Administration of phosphorodiamidate morpholino oligomer
Mice were anesthetized using 4 % isoflurane and 0.5 L/min 100 % oxygen and then maintained using 2 % isoflurane and 0.5 L/min oxygen delivered via a nose cone with a passive exhaust system on a warming device [34]. The phosphorodiamidate morpholino oligomer (PMO) mExon 23(+07-18) (5′- GGCCAAACCTCGGCTTACCTGAAAT- 3′) against the boundary sequences of exon and intron 23 of the mouse dystrophin gene was synthesized by Gene Tools (Philomath, OR, USA). PMO was administered via a single 800 mg/kg dose through an IV injection via the retro-orbital sinus as previously described [35]. PMO was administered in a volume of 300 μl in saline at an injection rate of 2 μl/s (2 min total injection time). After the injection, the mouse was placed back into its cage for recovery and monitored for pain or distress. Control mdx mice were injected with 300 μl saline exactly as described for the PMO-treated mice. Uninjected WT C57BL/10 mice were used as dystrophin-positive controls.
Tissue collection for various quantification methodologies
Mice were sacrificed 1 month after administration of PMOs. Mice were euthanized via carbon dioxide inhalation, and multiple muscle tissues were harvested (tibialis anterior, gastrocnemius, triceps, quadriceps, heart, and diaphragm) [36]. Muscle tissues were quickly removed surgically, cut into three parts, snap-frozen in liquid nitrogen-cooled isopentane, and stored at −80 °C for further analysis. For immunofluorescent staining, muscles were placed on cork, coated with OCT mounting medium, and frozen in liquid nitrogen-cooled isopentane.
Immunofluorescent staining
Dystrophin protein expression
Frozen muscle tissues were sectioned at 10-μm thick and stored at −20 °C until used. Immunofluorescent (IF) for dystrophin protein was performed as described previously [37]. In brief, the muscle sections were brought to room temperature (RT) but not fixed. For dystrophin staining, unfixed sections were blocked with 10 % normal sheep serum, followed by incubation overnight at 4 °C in a humidified chamber with a P7 dystrophin antibody (1:400; Fairway Biotech, England). The P7 antibody binds to the rod domain (exon 57) of the dystrophin protein. Next, the sections were washed and probed with goat anti-rabbit IgG Alexa 594 antibody (1:300; Life Technologies, Grand Island, NY, USA) at RT for 1 h and counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining. The stained tissue sections were stored at 4 °C for further imaging and quantification analyses. Staining was confirmed using alternative dystrophin antibody (Genetex, Irvine, CA, USA). Images were acquired using an Olympus BX61 microscope with attached Olympus DP71 camera module. The surface area of each section and the relative proportion of the dystrophin-positive fiber area were determined using ImageJ software.
Muscle fiber type
Muscle fiber types were identified using the following antibodies: mouse IgG2b monoclonal anti-type 1 MHC (clone BA-D5, 1:50), mouse IgG1 monoclonal anti-type 2a MHC (clone SC-71, 1:50), mouse IgM monoclonal anti-type 2b MHC (clone BF-F3, 1:5), and mouse IgG1 monoclonal anti-embryonic MHC (clone F1.652, 1:25), all obtained from the Developmental Studies Hybridoma Bank at the University of Iowa (Ames, IA, USA) [38]. Sections were double-stained with dystrophin antibody (Genetex).
In brief, serial cross sections (10-μm thick) were fixed in −20 °C acetone for 10 min. Sections were warmed to RT for 5 min and then incubated in phosphate-buffered saline (PBS) for 15 min, followed by a 1-h incubation in PBS with 0.5 % bovine serum albumin (BSA), 0.5 % Triton X-100, and 1 % horse/goat serum. After three 5-min washes with PBS, samples were incubated for 2 h with primary antibody. After three further 5-min washes with PBS with 0.1 % Tween-20, the samples were incubated for 1.5 h with secondary antibody at 1:500 dilution: Alexa 488-conjugated anti-mouse IgG Fc 2b (for type 1 fibers), Alexa 488-conjugated anti-mouse IgG Fc 1 (for type 2a and embryonic fibers), and Alexa 488-conjugated anti-mouse IgM (for type 2b fibers) (Invitrogen, Carlsbad, CA, USA). Samples were then washed three times for 10 min each, and the slides were mounted using Prolong Gold with DAPI (Life Technologies). Images were acquired using the Olympus BX61 VS virtual slide system (VS120-S5) with attached Olympus XM10 monochrome camera and Olympus VS-ASW FL 2.7 software.
Immunoblotting (IB) for dystrophin protein expression
Total protein was extracted from the frozen tissues (tibialis anterior, gastrocnemius, triceps, quadriceps, heart, and diaphragm muscles) using radioimmunoprecipitation assay buffer (RIPA) buffer (50 mm Tris-HCl, pH 8.0, with 150 mm sodium chloride, 1.0 % Igepal CA-630 (Nonidet P-40), 0.5 % sodium deoxycholate, and 0.1 % sodium dodecyl sulfate) (Teknova, Hollister, CA, USA) containing protease inhibitors (Halt protease inhibitor mixture 100X; Thermo Fisher Scientific, Waltham, MA, USA). Protein concentrations in the muscle lysates were estimated using the Bio-Rad Microplate Protein Assay (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s protocol.
Extracted proteins from mdx-saline (50 μg), mdx-PMO (50 μg), and C57BL/10 muscles (3.125 μg) were separated on a Tris-acetate 3–8 % gel (Life Technologies) and transferred overnight at 4 °C onto nitrocellulose membranes. Membranes were blocked using 5 % milk in TBS-Tween (0.1 % Tween) and incubated overnight at 4 °C with DYS1 and DYS2 monoclonal antibodies (1:1000; Leica Microsystems, Buffalo Grove, IL, USA). Membranes were then washed and probed with polyclonal rabbit anti-mouse HRP antibody (1:3000; DAKO, Carpinteria, CA, USA) for 1 h at room temperature. Next, the membranes were incubated with ECL Western Blotting Substrate (GE Healthcare, Piscataway, NJ, USA) and developed on X-ray film (Denville Scientific, South Planfield, NJ, USA). Similarly, membranes were probed with anti-vinculin (1:5000; Abcam Inc, Cambridge, MA, USA) and used as loading controls. Densitometric quantification of band intensity was measured using Quantity One software. The band area to be quantified was determined by identifying the area of the major dystrophin species band, which was kept constant between lanes for an individual blot for analysis. Any possible degradation products were not included in the quantification, as shown in Additional file 1B.
Dystrophin quantifications in morpholino-treated mdx muscle were calculated as follows:
percentage of dystrophin expression = (OD from mdx sample/OD from C57BL/10) × dilution factor = 16 (50 μg mdx/3.125 μg C57BL/10).
IB runs were performed three times. In the first run, the same amount of total protein was loaded for the WT and mdx samples (50 μg). For runs 2 and 3, WT samples were serially diluted to 3.125 μg total protein for loading.
Mass spectrometry for dystrophin protein expression
Dystrophin protein levels for the tibialis anterior, gastrocnemius, and triceps muscles were determined using MS for PMO-treated mdx mice (n = 6) in comparison to a C57BL/10 control, as described previously [39]. Using the same protein extracts as for the immunoblots, 50 μg of total protein for each muscle was mixed with 25 μg of an internal standard for stable isotope labeling of amino acid in mammals (SILAM) that had been extracted in the same RIPA buffer from a gastrocnemius muscle [40, 41]. A SILAM mouse is a C57BL/6J mouse fully labeled with 13C6-lysine, so that all lysine residues are 6 Da heavier [39, 40]. Unlabeled and labeled protein mixtures were separated by 1D electrophoresis. The region corresponding to approximately 300–500 kDa was excised and in gel-digested with trypsin. The resulting peptides were dried by vacuum centrifugation and resuspended in 20 μl of HPLC-grade water with 0.1 % formic acid and 2 % acetonitrile (buffer A). Each sample (5 μl) was injected onto a NanoEasy HPLC and loaded and equilibrated in Buffer A at 800 Bar onto an EasySpray C18 50 μm column, followed by a gradient of 0–35 % acetonitrile at 300 nL/min over 24 min, and coupled online to a Q Exactive mass spectrometer (ThermoFisher, San Jose, CA). The Q Exactive was operated in timed targeted MS2 mode for 13 unlabeled and labeled peptides with the following parameters: positive polarity; resolution 17,500; AGC 1e6; max IT 60 ms; MSX count 4; isolation width 2 m/z; first m/z 150; and NCE 27.
Timed targeted mass spectral data were analyzed using Skyline, version 2.6.0.6709 (skyline.gs.washington.edu) to determine the ratio of unlabeled to labeled for each transition for each peptide. A total of 13 dystrophin peptides and 3 filamin C peptides with four to seven y-ion transitions each were monitored. Peptides with poor co-elution transitions were removed (Skyline “Peptide Peak Found Ratio” score <0.9). Peptide ratios were averaged to give the mean protein ratio. The dystrophin ratio was compared to the filamin C ratio for each sample. PMO-treated samples were compared to the corresponding C57BL/10 muscle to determine the percentage of normal.
ELISA for PMO quantification in muscle lysates
Protein lysates from the previous dystrophin quantification experiments by IB and MS were used for PMO quantification by a high-sensitivity hybridization ELISA, as previously described [42]. In brief, sample lysates were diluted 1/20, 1/200, and 1/2000 in a control muscle lysate buffer (0.2ug/μL protein), and PMO standards were diluted to various concentrations in a similar manner. Hybridization was facilitated using an anti-sense probe to the PMO with both a biotin epitope and a DIG tag for hybridization and carried out at 37 °C. After hybridization, 100 μL of the hybridization mix was pipetted into duplicate wells on avidin-coated plates and incubated at 37 °C for 30 min. The plates were then washed, and each well was treated with micrococcal nuclease (NEB, M02475), followed by incubation with an anti-digoxigenin-AP Fab fragment antibody (Roche, 11093274910). Lastly, AttoPhos Substrate (Promega, S101C) was added to the plates and incubated at 37 °C for 30 min. Fluorescent readings were obtained, and PMO concentrations were quantified and calculated on the basis of the PMO standard curve.
Statistical analysis
All data are presented as dystrophin percent of C57BL/10 (normal) and means ± standard deviation of the mean. Correlation analysis between quantification methods was performed to determine Spearman’s statistical correlations. p < 0.05 was considered significant.