Female WT (C57BL/6J) and mdx
mice (C57BL/10ScSnJ) were purchased from Jackson Laboratories (Bar Harbor, ME, USA). Male Akt DTG mice possessed the 1256
transgene expressing the reverse-tetracycline transactivator controlled by a mutated skeletal MCK promoter and the TRE-myrAkt1
transgene harboring the constitutively active form of the mouse Akt1
transgene controlled by a tetracycline-responsive promoter (29
). Female WT and mdx
mice were bred with founder Akt DTG males to produce the four genotypes used for comparison: (i) WT with a single transgene (WT STG), (ii) WT with both transgenes (WT DTG), (iii) mdx
with a single transgene (mdx
STG) and (iv) mdx
with both transgenes (mdx
DTG). Only male progeny were used in experiments.
For mdx-Akt transgenic comparisons, mice were treated starting at 6 weeks of age with 0.5 mg/ml DOX administered in drinking water. Following 3 weeks of treatment, mice were euthanized via inhalation of isoflurane anesthetic and tissues (blood, quadriceps, EDL, total skeletal muscle) were harvested. For CTX experiments, mice were treated with DOX at 12 weeks of age and sacrificed between 14 and 16 weeks of age (see below). Experimental procedures and animal maintenance were conducted with the approval of the Institutional Animal Care and Use Committee (IACUC) at UCLA.
Genotyping of Akt transgenic and mdx mice
Genomic DNA was isolated from mouse tail clippings using the DNeasy Blood and Tissue Kit (QIAGEN Inc., Valencia, CA, USA; #69506). Polymerase chain reaction (PCR) genotyping of WT and mdx
dystrophin alleles was performed through the modified amplification-resistant mutation system (ARMS) assay (67
). The MCK-rtTA
transgenes were identified using PCR through previously described methods (37
Quadriceps were dissected, weighed, mounted in optimal cutting temperature (OCT) tissue freezing medium (10.2% polyvinyl alcohol/4.3% polyethylene glycol), frozen in liquid nitrogen-cooled isopentane and stored at −80°C. For histological analyses, quadriceps were sectioned transversely at 8 µm in a CM 3050S cryostat (Leica Microsystems, Bannockburn, IL, USA) and mounted on Superfrost Plus positively charged slides (VWR International, West Chester, PA, USA; #48311-703). H&E staining was used for visualization of fibrosis, central nucleation and cross-sectional fiber area as previously described (37
). Centrally nucleated fibers and cross-sectional fiber area were measured from digitized images captured with the AxioPlan 2 fluorescent microscope and AxioVision 4.8 software (Carl Zeiss Inc., Thornwood, NY, USA). Central nucleation was quantified as a percentage of centrally nucleated fibers over the total number of fibers in an entire transverse quadriceps section. Cross-sectional areas of fibers were sampled from 300 adjacent fibers in each quadriceps and calculated using the outline spline function.
EBD tracer assay
To measure sarcolemmal permeability, 50 µl per 10 mg of body weight of sterile EBD (10 mg/ml in PBS) was intraperitoneally injected into mice 18 h before tissue harvesting. For visualization of EBD-infiltrated myofibers in the quadriceps, cryosections were handled in the dark. Sections were fixed in ice-cold acetone prior to blocking with 3% bovine serum albumin and incubation with an anti-laminin primary antibody (Sigma, St Louis, MO, USA; #L 9393), which was detected with Alexa Fluor 488 (Invitrogen Corporation, Carlsbad, CA, USA; A11008, 1:200 dilution) labeled anti-rabbit antibody. Sections were mounted in VectaShield (Vector Laboratories, Burlingame, CA, USA; H-1000) and imaged using the AxioPlan 2 fluorescent microscope and AxioVision 4.8 software. Whole quadriceps mosaics were photographed, and sarcolemmal integrity was quantified through the percentage of EBD-positive fibers as a percentage of total fibers counted in the section.
Total skeletal muscles were collected, snap frozen in liquid nitrogen and stored at −80°C prior to use. Prior to protein lysate preparation, frozen skeletal muscles were crushed in liquid nitrogen with a mortar and pestle. Ice-cold RIPA lysis buffer (Thermo Scientific, Rockford, IL, USA; #89901) was modified by adding phosphatase inhibitors [1 mm sodium orthovanadate, 100nm okadaic acid and 5nm microcystin LR) and protease inhibitors (0.6 µg/ml pepstatin A, 0.5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.75 mm benzamidine and 0.1 mm phenylmethylsulfonyl fluoride PMSF)]. Ten milliliters of modified RIPA buffer were added per gram of pulverized muscle, which was homogenized using a tissue miser at the lowest speed (Fisher Scientific, Pittsburgh, PA, USA). Homogenates were rotated for 1 h at 4°C, and centrifuged at 15 000g at 4°C for 15 min, after which the clarified supernatants were removed for use. Protein concentrations were measured using the DC Protein Assay (Bio-Rad, Hercules, CA, USA; #500-0111). Equal concentrations of protein (60 µg) were resolved through 4–20% gradient SDS–PAGE (Pierce, Rockford, IL, USA) and transferred to nitrocellulose membranes (Millipore, Billerica, MA, USA) for immunoblotting experiments.
Membranes were probed with antibodies to the following proteins, listed with their working dilutions: dystrophin [Developmental Studies Hybridoma Bank (DSHB), Iowa City, IA, USA; MANDYS, 1:10], utrophin (DSHB; MANCHO3, 1:5), α-DG (Millipore; IIH6, 1:700), β-DG (DSHB; MANDAG2, 1:20), α-SG (Vector Laboratories; VP-A105, 1:100), γ-SG (Vector Laboratories; VP-G803, 1:200), β1D integrin (Chemicon International, Temecula, CA, USA; MAB1900, 1:200), nNOS (Invitrogen; #61-7000, 1:400), dysferlin (Abcam Inc., Cambridge, MA, USA; ab55988, 1:600), Akt (Cell Signaling Technologies, Beverly, MA, USA; #9272, 1:500), phosphorylated Akt (Ser 473, Cell Signaling Technologies; #9271, 1:500), HA tag (Sigma; H 3663, 1:5000) and GAPDH (Chemicon International; MAB374, 1:30,000). All primary antibodies were detected using horseradish peroxidase-conjugated secondary antibodies directed against mouse (GE Healthcare, Piscataway, NJ, USA; NA931V, 1:3,000), rabbit (GE Healthcare; NA934V, 1:3,000) or goat (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; SC-2033, 1:3000) IgG. Immunoblots were developed using SuperSignal West Pico Chemiluminescent Substrate (Pierce; #34080). Relative changes in protein levels were quantified by densitometry analysis of immunoblot bands using the Alpha Imager 2200 and Alpha Imager v5.5 software (Alpha Innotech, Santa Clara, CA, USA).
Indirect immunolabeling experiments using mouse monoclonal antibodies were conducted in combination with the Vector M.O.M Immunodetection Kit (Vector Laboratories; BMK-2202) according to the manufacturer's protocol. For α-DG staining, sections were fixed in pre-chilled 50% ethanol plus 50% acetic acid for 1min prior to washing and blocking steps. Transverse cryosections were incubated overnight with mouse antibodies to detect the following proteins, listed with their working dilutions: dystrophin (1:2), utrophin (1:5), α-DG (Millipore; VI A4-1, 1:40), β-DG (Vector Laboratories; BP-B205, 1:25), α-SG (1:30), β-SG (1:30), γ-SG (1:30), sarcospan [Rabbit 3 (69
), 1:5], β1D integrin (1:25), nNOS (1:100) and dMHC (Novocastra, Newcastle upon Tyne, UK; NCL-MHCd, 1:40). Primary antibodies were detected via incubation with biotinylated secondary antibody against mouse (Vector Laboratories; BA-9200, 1:250) or rabbit (Vector Laboratories; BA-1000, 1:250) followed by fluorescein Avidin D (Vector Laboratories; A-2001, 1:250). Sections were mounted in VectaShield and imaged using the AxioPlan 2 fluorescent microscope and AxioVision 4.8 software. Images were taken under identical conditions so that appropriate comparisons could be made between the four different genotypes.
Forelimb grip strength measurements
One day prior to sacrifice, DOX-treated 9-week-old mice were subjected to forelimb grip strength tests using a horizontally positioned grip strength meter (Columbus Instruments, Columbus, OH, USA; DFIS2 Chatillon CE). Mice were lowered by the tail towards the metal pull bar on the apparatus. Upon grasping the bar with their forelimbs, mice were then pulled backwards in the horizontal plane. The procedure was repeated consecutively five times and the peak tension (N) of the five pulls was recorded as the grip strength value. Each animal was subjected to a total of five serial trials of five pulls each with 30 s of rest in between trials.
In vitro contraction force measurements
Mice were euthanized with isoflurane and the EDL muscles were quickly dissected out while being superfused with chilled mammalian Ringer's solution. One tendon was fixed to the bottom of a recording chamber, while the other tendon was attached to a Dynagage DG-600D capacitive tension transducer (Whittaker, North Hollywood, CA, USA). The chamber was perfused with room temperature (~22°C), oxygenated mammalian Ringer containing: 119 mm NaCl, 5 mm KCl, 1 mm MgSO4, 5 mm NaHCO3, 1.25 mm CaCl2, 1 mm KH2PO4, 10 mm HEPES, 10 mm dextrose and 70 µl/100 ml insulin–transferrin–selenium A (Gibco; # 51300-044), with 30 µM d-tubocurarine chloride to block neuromuscular transmission. Direct muscle stimulation was achieved by passing current pulses between Pt plate electrodes placed on either side of the muscle. The muscle was held at the resting length at which twitch tension was maximal. Stimuli of 15–30 V were produced by a Grass S4 stimulator, driven by a computer-triggered Nihon Kohden stimulator that set the pulse duration (0.5 ms) and repetition frequency (300 ms trains of pulses at 1, 10, 20, 40, 60, 80, 100 and 150 Hz), repeated at 30 s intervals of 1–20 Hz, 2 min intervals at 30–60 Hz and 5 min intervals at >80 Hz. The output of the tension transducer was digitized at a 10 kHz sampling rate by a Digidata 1200A A/D converter and stored and analyzed with Clampfit 9.2 software (Axon Instruments). After a full recovery, fatigue was tested by 150 repetitions of tetanic stimulation at 100 Hz for 300 ms every 2s, totaling for 300 s. The entire recording sequence normally was completed in ~90 min. The right EDL from each mouse was held in the same recording chamber and tested in the same way after completion of the measurements on the first, left EDL muscle. Whereas both left and right muscles usually gave similar results, data from the side corresponding to the greater maximum tension were used.
For CTX experiments, 12-week-old male WT Akt transgenic mice were treated with 0.5 mg/ml DOX in drinking water. After 2 weeks of treatment, mice were anesthetized with isoflurane and were shaved on the hindlimbs for visualization of the quadriceps. CTX from N. nigricollis (CTX, 10 µm in PBS; EMD Chemicals, Gibbstown, NJ, USA; 217504) at a volume of 200 µl was injected deep into the quadriceps muscle, and 200 µl of PBS was injected into the contralateral quadriceps as a control. Mice were continually treated with DOX throughout injury recovery, and quadriceps were harvested at 1, 2, 4 and 7 days following injection, as well as day 0 prior to an injection.
For histological analysis, the entire quadriceps was surveyed at five levels from proximal to distal regions. For each level, 8μm transverse cryosections were collected in replicates of 50, followed by a collection of 1 mm of quadriceps tissue not used for histological analysis. Representative images from H&E staining, EBD fluorescence and dMHC indirect immunofluorescence reveal areas of the quadriceps with the maximum histological damage. To achieve dual label imaging of dMHC and laminin, tissue sections were extensively photobleached to eliminate EBD fluorescence prior to staining with laminin and dMHC antibodies.
For quadriceps mass, each data point was presented as the average mass of both left and right quadriceps of each animal. For central nucleation and EBD quantification, values from individual quadriceps were treated independently, because of variance in pathology observed within mdx animals. Statistical significance for all studies (with the exception of muscle strength time trials) was determined through two-way analysis of variance (ANOVA) on ranks. Two-way ANOVA was conducted in a 2 × 2 factorial design, with dystrophin genotype (WT and mdx) as the first factor and Akt transgene genotype (STG and DTG) as the second factor. Serial grip strength trials were compared using three-way mixed ANOVA with repeated measures to identify decline in strength. The two factors examined were dystrophin genotype and transgene genotype, with the third factor being the five trials within subjects. Fatigue measurements in mdx STG and DTG EDL muscles were analyzed using mixed model ANOVA with the factors of time as a continuous variable and genotype as a categorical variable. Post hoc pairwise comparisons were made using Student's t-test with Bonferroni correction using a familywise α-level of P< 0.05.