Animals

Mice were group-housed with access to food and water ad libitum and under a 12 h light/dark cycle (lights on at 7:00 A.M.) at a temperature of 22 ± 1 °C. For MRI study, male mdx mice (C57BL/10ScSn-Dmdmdx/J, 001801, n = 8) and age-, sex-matched wild-type (WT) mice (C57BL/10ScSnJ, 000476, n = 8) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). For EIM studies, male mdx mice (C57BL/10ScSn-Dmdmdx/JicJcl, n = 43) and WT mice (C57BL/10Sn Slc, n = 33) were purchased from CLEA Japan (Tokyo, Japan) and Japan SLC (Hamamatsu, Japan), respectively. For the natural history study of EIM measurements and histopathology, mice were allocated randomly. EIM was continuously recorded from the same individual (n = 6 for WT, n = 6 for mdx mice) at 6, 12, 18, and 24 weeks of age, with muscle tissue sampled for histopathology at 24 weeks of age after all the EIM recordings. Additionally, for histopathology, muscle tissue was sampled after EIM recording at 6, 12, or 18 weeks of age (n = 5 or 6/group/time point). For the drug intervention study using EIM, male mdx mice (n = 20) and WT mice (n = 10) at 14–15 weeks of age were used. All experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Shonan Health Innovation Park or Northeastern University and were conducted in accordance with the guidelines.

MRI and magnetic resonance spectroscopy (MRS)

These experiments were performed at the Northeastern University (Boston, MA, USA). Mice were initially anesthetized with isoflurane (5% for induction and 2% during setup) and then secured in a prone position on a Bruker cradle. During the study, respiration rate was monitored using a balloon secured under the mouse’s chest and a pressure transducer. Body temperature was measured with a rectal probe and maintained at ~ 36 °C with a feedback-controlled air heater (SA Instruments, Inc., Stony Brook, NY, USA). All MRI measurements were performed using a Bruker Biospec 7.0-T, 20 cm bore USR horizontal system (Bruker Scientific, Billerica, MA, USA). An actively decoupled 2 cm diameter surface coil positioned beneath the thighs/legs of the mouse was used as the radiofrequency (RF) receiver, and an actively decoupled 72 mm diameter volume coil was used as the RF transmitter. Magnetic field homogeneity was optimized using automatic global shimming. Pilot images in three directions (axial, sagittal, and coronal) were acquired using a fast, low-angle shot (FLASH) sequence. A multi-slice multi-echo (MSME) sequence was used for T2 measurement [27,28,29]. T2 MRI data were acquired from 16 consecutive axial slices with a slice thickness of 1.5 mm, which covered the thighs and legs. The MRI parameters for the MSME sequence were as follows: matrix = 128 × 128, field of view = 25 × 25 mm2, in-plane resolution = 0.2 × 0.2 mm2, repetition time (TR) = 4 s, first echo time (TE) = 11 ms, 10 echoes with 11 ms echo spacing, no averaging, and total acquisition time = 512 s. Previous studies have reported that factors such as stimulated echoes can cause errors in T2 measurement due to the contiguous slices and/or multiple echoes [30, 31]. The Bruker MSME protocol adopts two mechanisms to minimize such errors. First, the MRI data in the sixteen continuous slices were acquired with “interleaved” scheme (i.e., the sequence to acquire the data from the 16 contiguous slices is 1 → 3 → 5 ··· 15, 2 → 4 → 6 ··· 16), which introduces an actual interslice gap of 100% to slice thickness. Secondly, spoiling gradients were applied on both sides of the refocusing RF pulses in Slice and Read directions, which would eliminate the unwanted stimulated echoes and minimize the errors in T2 measurement [30, 31]. The reliability of the Bruker MSME protocol for T2 measurement has been validated by both phantom and in-vivo studies [27]. The T2 maps were calculated using Bruker’s Paravision 6.0.1. MRI images and T2 data were analyzed using ImageJ software (https://imagej.nih.gov/ij/). For T2 measurement, oval regions of interest (ROI) of the same size were selected across three consecutive slices in each leg close to the knee bones (Supplementary Fig. 1A). The area of each ROI was 160 pixels (6.1 mm2), and they avoided fibula, larger blood vessels, and the subcutaneous fat, and they covered the posterior part of the hindlimb which mainly include gastrocnemius, soleus, and plantaris muscles as shown in the Supplementary Fig. 1B. Because there was no difference in tibia bone volumes between mdx and WT mice [32], leg muscle size was measured as the average cross-sectional area (CSA), without subtracting the tibia bone regions in these three consecutive slices.

A single-voxel stimulated echo acquisition-mode (STEAM) MRS sequence was used to measure the fat fraction in the muscles [33, 34]. A voxel of size 2 × 2 × 2 mm3 was located in the posterior muscles of the leg for MRS. Localized shimming provided by Bruker was used to minimize the spectral linewidth. The parameters for STEAM were as follows: TR = 2 s, TE = 3 ms, acquisition bandwidth = 10,000 Hz, and acquisition data points = 2048. The routine MRS signal was dominated by the water signal, and an average number of 1 achieved a high signal-to-noise ratio. To enhance the fat signal, the same parameters as the water MRS measurement were used, but variable pulse power and optimized relaxation delay (VAPOR) water suppression was added to the STEAM sequence [35]. To improve the signal-to-noise ratio, the number of averages was increased to 64. The fat fraction was calculated as the fat/water ratio by dividing the magnitude of the fat peak (located at 1000 Hz) by that of the water peak (located at 0 Hz) in the water spectrum (Supplementary Fig. 2) [36].

EIM measurements

EIM data were obtained from mice placed on a heating pad to maintain consistent body temperature under 1.5% isoflurane anesthesia. The left hindlimb was taped to the measuring surface at an approximately 45° angle extending out from the body. After shaving the hindlimb far and removing the remaining hair with a depilatory cream (Cha.lu.la Disahair cream, GARDEN, Osaka, Japan), calf thickness was measured using a dial thickness gauge (Peacock G-2; Ozaki MFG Co. Ltd., Tokyo, Japan). The skin at the measurement site was pretreated with a skin preparation gel (Nuprep®; Weaver and Company, Aurora, CO, USA) to reduce skin impedance. A fixed rigid four-electrode array with a 20 g weight attached to the tip was placed over the gastrocnemius (GC) muscle in the longitudinal direction [37] after applying an electrode cream (SignaCreme®; Parker Labs, Fairfield, NJ, USA), which facilitates electrical conduction between the skin and electrodes. A weak electrical current with a wide range of frequencies was applied to the tissues through one set of electrodes, and the resulting voltages were measured via a second set. EIM measurements were performed using an impedance spectroscopy system (mScan-V™; Myolex Inc., Boston, MA, USA) to obtain three EIM parameters: reactance (X), resistance (R), and phase (arctan X/R). Multi-frequency measurements over the 1–10,000 kHz range within several seconds were repeated three times and averaged. For the analyses, values from 10 to 1000 kHz were used to avoid both low- and high-frequency artifacts.

Skeletal muscle histopathology

Mice were anesthetized by intraperitoneal injection of a combination anesthetic (medetomidine 0.3 mg/kg, midazolam 4 mg/kg, and butorphanol 5 mg/kg) and sacrificed by intracardiac perfusion initially with saline, followed by a fixation with 4% paraformaldehyde phosphate buffer solution (163–20145; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) The hindlimbs including GC muscles were removed and immersed in the same fixative for 24 h at 4 °C. The fixative was then replaced with 0.1 mol/L phosphate buffer solution (pH 7.4) and stored at 4 °C until the dissection of hindlimb GC muscles. The GC muscle tissues were sectioned from the left hindlimb, in which the EIM was measured, and embedded in paraffin for histological analysis. Tissue Sects. (4 μm thick) were stained with hematoxylin and eosin (H&E) for histopathological analysis. The slides were evaluated by a board-certified veterinary pathologist.

To determine the muscle fiber size (CSA) and count, laminin immunohistochemical staining was performed using a BOND RX automated stainer (Leica Biosystems, Deer Park, IL, USA). Transverse sections of muscles (4 μm thick) were stained for the cell membrane with a rat monoclonal anti-laminin 2 alpha antibody (4H8-2, ab11576, dilution 1:1000; Abcam, Cambridge, UK) for 30 min at room temperature. The cell membrane of the muscle fibers was visualized with Polink-2 Plus HRP Rat-NM DAB Detection Kit (D46-6; OriGene Technologies, Inc., Rockville, MD, USA), the Bond Polymer Refine Detection Kit (DS9800; Leica Biosystems, Deer Park, IL, USA), and DAB Enhancer (AR9432; Leica Biosystems, Deer Park, IL, USA). Subsequently, the sections were stained with hematoxylin, dehydrated with 100% ethanol, and mounted using Permount Mounting Medium (SP15-100; Thermo Fisher Scientific, Waltham, MA, USA). Digital images were obtained using a digital slide scanner (NanoZoomer S60; Hamamatsu Photonics, Shizuoka, Japan), and the CSA and muscle fiber count were subsequently analyzed using the image analysis software HALO (Indica Labs, Albuquerque, NM, USA). The analyzed muscle fibers were categorized into three types based on their thickness (small: < 500 μm2, middle: 500–1500 μm2, large: > 1500 μm2) to characterize genotype-related differences.

To evaluate the fibrotic area in the muscles, Sirius red (SR) staining was performed using an automated stainer (DRS2000; Sakura Finetek USA, Inc., Torrance, CA, USA). Dewaxed muscle transverse Sects. (4 μm thick) were stained for collagen with a modified Sirius red staining solution, which consisted of Sirius Red (0.1% w/v, 196–16201; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), Fast Green FCF (0.1% w/v, 069–00032; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), and Van Gieson Solution P (224–01405; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan), for 30 min at room temperature. Subsequently, the sections were rinsed with deionized water, dehydrated with 100% ethanol, and mounted using Permount Mounting Medium (SP15-100; Thermo Fisher Scientific, Waltham, MA, USA). Images of the stained sections were captured using a NanoZoomer S60 (Hamamatsu Photonics, Shizuoka, Japan), and areas (μm2) of SR-positive and total tissues were analyzed using HALO (Indica Labs, Albuquerque, NM, USA). The SR-positive area (%) was calculated as a measure of the extent of fibrosis excluding the fascia, large tendons, blood vessels, and peripheral nerves from the total tissue area.

Treatment with a peptide-conjugated PMO

A peptide Pip9b2 (Ac-RXRRBRRFQILYRBRXRB-OH, where ‘X’ was aminohexanoic acid, and ‘B’ was β-alanine)-conjugated PMO (PPMO) [25] that was designed to induce skipping of Dmd exon 23 in mice, was manufactured at Takeda Pharmaceutical Company Limited. PPMO was dissolved in saline at a concentration of 2 mg/mL and then warmed for 10 min at 65 °C followed by vortex mixing.

Twenty mdx mice at 15 weeks of age were used for pre-EIM recordings. Based on the EIM data, one individual was excluded from the analysis due to exceeding 2 standard deviations (SD) from the mean and then the remaining mdx mice were divided into two groups (n = 9 for vehicle-treated group, n = 10 for PPMO-treated group). Thereafter, the mdx mice were intravenously administered PPMO (10 mg/kg) or saline via the tail vein four times every two weeks, starting at 16 weeks of age. To evaluate the drug effect, EIM was recorded twice, at 2 weeks after the first and fourth injections (2W post-dose at 18 weeks of age and 8W post-dose at 24 weeks of age). After completing the 8W post-dose EIM recordings, the animals were sacrificed to obtain GC muscle samples for exon skipping efficiency analysis and dystrophin protein quantification. The samples were stored at -80 °C until use.

Real-time quantitative polymerase chain reaction (RT-qPCR) and the calculation of exon skipping efficiency

GC muscle tissues were homogenized using a FastPrep-24 5G homogenizer (MP Biochemicals LLC, Irvine, CA, USA) in Lysing Matrix I tubes (6918–100; MP Biochemicals LLC, Irvine, CA, USA) filled with ISOGEN (319–90211; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan). Total RNA was extracted using phenol/chloroform extraction, followed by QuickGene RNA Tissue Kit SII (RT-S2; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) according to the manufacturer’s instructions. The concentration of total RNA was measured using a microvolume UV–Vis spectrophotometer (NanoDrop 8000; Thermo Fisher Scientific, Waltham, MA, USA) and cDNA was synthesized from 200 ng of total RNA using a High-Capacity cDNA Reverse Transcription Kit (4368813; Invitrogen, Carlsbad, CA, USA) in 20 μL of total reaction volume. RT-qPCR was performed using QuantStudio (Thermo Fisher Scientific, Waltham, MA, USA) and Taqman Gene Expression Master Mix (4369016; Thermo Fisher Scientific, Waltham, MA, USA). One microliter of cDNA solution was used as the template in a 10 μL PCR reaction. The following primers were used for the mouse Dmd mRNA containing exons 22, 23, and 24 (non-skipped):

Forward primer, 5’-CCAAGAAAGCACCTTCAGAAATA-3’;

Reverse primer 5’-AGGAAAGTTTCTTCCAGTGC-3’; and

TaqMan probe, 5’- TCTGTCAGAATTTGAAGAGATTGAGGG-3’.

The following primers were used for mouse Dmd mRNA containing exons 22 and 24 (exon 23 skipped):

Forward primer, 5’-CTCTCTGTACCTTATCTTAGTGTTACTG-3’;

Reverse primer 5’-GGCAGGCCATTCCTCTTT-3’; and

TaqMan probe, 5’-CTCGGGAAATTACAGAATCACATAAAAACCT-3’.

The following primers were used for mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH):

Forward primer, 5’-CCCTGTTCCAGAGACGGCC-3’;

Reverse primer 5’-GCCAAATCCGTTCACACCGA-3’; and

TaqMan probe, 5’-CAGTGCCAGCCTCGTCCCGTAGACAAA-3’.

The expression levels of exon 23 non-skipped and exon 23 skipped were normalized to GAPDH expression. The percentage of exon 23 skipping efficiency was calculated using the following formula: expression level of exon 23 skipped / (expression level of exon 23 skipped + expression level of non-skipped) × 100.

Dystrophin protein analysis

GC muscle tissues were homogenized using a FastPrep-24 5G homogenizer (MP Biochemicals LLC, Irvine, CA, USA) in Lysing Matrix I tubes (6918–100; MP Biochemicals LLC, Irvine, CA, USA) with radioimmunoprecipitation assay (RIPA) buffer (182–02451; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) containing a protease inhibitor cocktail (P8340; Sigma-Aldrich Corp., Saint Louis, MO, USA) and 5 mM EDTA (347–07481; FUJIFILM Wako Pure Chemical Corp., Osaka, Japan) (modified RIPA buffer). After a 30 min incubation on ice, the homogenates were transferred to new tubes and centrifuged at 12,000 rpm for 20 min at 4 °C. The supernatants were collected and the total protein concentration was adjusted to 4 mg/mL. Dystrophin expression was analyzed with capillary electrophoresis immunoassay using the Wes system (ProteinSimple, San Jose, CA, USA), 66–440 kDa Wes Separation Module (SM-W008; ProteinSimple, San Jose, CA, USA), anti-rabbit detection module (DM-001; ProteinSimple, San Jose, CA, USA), and anti-Dystrophin antibody (ab154168; Abcam, Cambridge, UK) as previously reported [38]. Samples diluted with modified RIPA buffer were loaded into capillaries at a concentration of 0.2 mg/mL of total protein. To calculate the dystrophin expression levels, a standard curve was generated using a mixture of WT mice samples diluted with modified RIPA buffer at a range of concentrations from 0.025 to 0.4 mg/mL of total protein (12.5–200% of dystrophin levels in WT mice). Dystrophin expression values were plotted along a standard curve and calculated as the percentage of dystrophin in the WT mice.

Data analysis

Statistical analyses were performed using SAS software (SAS Institute Japan, Tokyo, Japan). To compare age-related changes between WT and mdx mice, data were assessed using Student’s t-test with a closed testing procedure. The comparison was performed at the endpoint at which the maximum difference was predicted. If the resulting two-sided p-value was < 0.05, the same comparison was repeated retrospectively to determine the earliest time point with a significant difference. The relationships between 50 kHz EIM reactance and histological parameters (averaged CSA, small fiber frequency, and fibrosis area) were assessed using Pearson’s correlation coefficient. In the drug intervention study, the effect of PPMO in mdx mice was evaluated using Student’s t-test with a closed testing procedure. Regarding 50 kHz EIM data, if a significant difference was detected between vehicle-treated WT and mdx mice, the comparison between vehicle-treated and PPMO-treated mdx mice was assessed. To compare the effects between single and repeated treatments, the above analysis was performed using 8W post-dose EIM data, in which the maximum treatment effect was expected. If a significant effect was detected in 8W post-dose EIM, 2W post-dose EIM data were subsequently analyzed. Statistical significance was set at p < 0.05. Data are presented as the mean ± standard error of the mean (SEM).