Figure 3.

Glycine Promotes PMO Uptake in Synergizing with Muscle Regeneration in mdx Mice

(A) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in mdx TA muscles injected with 2 μg of PMO followed by separate administration of glycine 16 h later (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). Red staining on fiber membrane shows dystrophin expression. Nuclei were counterstained with DAPI (blue) (the same is true for the rest unless otherwise specified). (B) Western blot and quantitative analysis for the dystrophin protein in treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (C) Immunohistochemistry for PAX7⁺ and Ki67⁺ muscle satellite cells (MuSCs) in TA and gastrocnemius muscles from mdx mice treated with saline or glycine every other day for 1 week intravenously (scale bar, 100 μm). TA, tibialis anterior. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (D) Quantitative analysis for PAX7⁺ and PAX7⁺/Ki67⁺ MuSCs in TA and gastrocnemius muscles from treated mdx mice (n = 4; two-tailed t test). (E) Immunohistochemistry for embryonic myosin heavy chain -positive (eMyHC⁺) regenerating myofibers in gastrocnemius from treated mdx mice. FITC-labeled PMO in glycine (PMO-G) or saline (PMO-S) was intravenously administered into adult mdx mice at 50 mg/kg for single injection and muscles were harvested 48 h later (scale bar, 100 μm). Fluorescently tagged wheat germ agglutinin (WGA) was used for the visualization of connective tissues. The arrowheads point to the eMyHC⁺ regenerating myofibers and FITC-labeled PMO. (F) Quantitative analysis for the fluorescence intensity of eMyHC⁺ regenerating myofibers in TA and gastrocnemius muscles from treated mdx mice (n = 3; two-tailed t test). (G) Representative RT-PCR to detect the exon-skipping efficiency, which is shown by shorter exon-skipped bands (indicated by Δexon 23, exon 23 skipped). G, gastrocnemius. (H) Measurement of the uptake of FITC-labeled PMO in differentiating and proliferating myoblasts treated with different concentrations of glycine (n = 4; one-way ANOVA and post hoc Student-Newman-Keuls test). NC refers to untreated differentiating myotubes or proliferating myoblasts. (I) Tissue distribution of FITC-labeled PMOs in mdx mice and quantitative analysis of fluorescence intensity in body-wide tissues. Body-wide tissues were harvested 48 h after single intravenous injection of FITC-labeled PMO in glycine (PMO-G) or saline (PMO-S) at the 50 mg/kg doses (n = 3; two-tailed t test). A, abdominal muscle; Q, quadriceps; TA, tibialis anterior; G, gastrocnemius; T, triceps; H, heart; K, kidney; L, liver; B, brain. Data are presented as means ± SEM. ∗p < 0.05.