Glycine Enhances Satellite Cell Proliferation, Cell Transplantation, and Oligonucleotide Efficacy in Dystrophic Muscle

Abstract

The need to distribute therapy evenly systemically throughout the large muscle volume within the body makes Duchenne muscular dystrophy (DMD) therapy a challenge. Cell and exon-skipping therapies are promising but have limited effects, and thus enhancing their therapeutic potency is of paramount importance to increase the accessibility of these therapies to DMD patients. In this study, we demonstrate that co-administered glycine improves phosphorodiamidate morpholino oligomer (PMO) potency in mdx mice with marked functional improvement and an up to 50-fold increase of dystrophin in abdominal muscles compared to PMO in saline. Glycine boosts satellite cell proliferation and muscle regeneration by increasing activation of mammalian target of rapamycin complex 1 (mTORC1) and replenishing the one-carbon unit pool. The expanded regenerating myofiber population then results in increased PMO uptake. Glycine also augments the transplantation efficiency of exogenous satellite cells and primary myoblasts in mdx mice. Our data provide evidence that glycine enhances satellite cell proliferation, cell transplantation, and oligonucleotide efficacy in mdx mice, and thus it has therapeutic utility for cell therapy and drug delivery in muscle-wasting diseases.

Keywords: Duchenne muscular dystrophy; cell therapy; exon skipping; glycine; mTORC1; satellite cell.

Graphical abstract

Figure 1

Screening of Amino Acids with PMO in Adult mdx Mice (A) Different concentrations of nutrients used for the study. (B–E) Dystrophin expression following single intramuscular injection of 2 μg of PMO into mdx TA muscles (B and C) or intravenous injection of PMO at 25 mg/kg/week for 3 weeks in different amino acid solutions or saline in mdx mice, respectively (D and E). (B) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in mdx TA muscles treated with single intramuscular injection of 2 μg of PMO in different nutrients (scale bar, 100 μm). The concentration was 5%, as illustrated in (A). For glycine and saline groups, the number of animals used per group is six (n = 6) and the rest is three (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). White staining on fiber membrane shows dystrophin expression. (C) Representative western blot and quantitative analysis for dystrophin expression in TA muscles from mdx mice treated with single intramuscular injection of PMO in different solutions. For glycine and saline groups, the number of animals used per group is six (n = 6) and the rest is three (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). α-Actinin was used as the loading control. 5, 2.5, and 0.5 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. TA muscles from C57BL/6 mice were used as normal controls (the same is true for all western blots unless otherwise specified). (D) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated intravenously with PMO in saline or amino acid solutions (5%) at 25 mg/kg/week for 3 weeks. TA, tibialis anterior. Scale bar, 100 μm. (E) Western blot and quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated intravenously with PMO in glycine (n = 4), serine, or saline (n = 3) at 25 mg/kg/week for 3 weeks (one-way ANOVA and post hoc Student-Newman-Keuls test). 0.5, 2.5, and 5 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. (F) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated intravenously with PMO in glycine at 25 mg/kg/week for 3 weeks with glycine (+Gly) or without additional glycine (−Gly) every other day for 5 weeks (scale bar, 100 μm). (G) Western blot for dystrophin expression in body-wide muscles from mdx mice treated intravenously with PMO in glycine at 25 mg/kg/week for 3 weeks with glycine (+Gly) or without additional glycine (−Gly) every other day for 5 weeks. 0.5, 2.5, 5, and 15 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. TA, tibialis anterior; Q, quadriceps; G, gastrocnemius; T, triceps; A, abdominal muscle; D, diaphragm. (H) Quantitative analysis of dystrophin expression in body-wide muscles from mdx mice treated intravenously with PMO in glycine at 25 mg/kg/week for 3 weeks with glycine (+Gly) or without additional glycine (−Gly) every other day for 5 weeks (n = 4; one-way ANOVA and post hoc Student-Newman-Keuls test). (I) ELISA assay for measurement of glycine in serum from mdx mice treated intravenously with PMO in glycine at 25 mg/kg/week for 3 weeks with glycine (+Gly) or without additional glycine (−Gly) every other day for 5 weeks (n = 4). Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.

Figure 2

Long-Term Repeated Administrations of PMO in Glycine (PMO-G) or in Saline (PMO-S) in Adult mdx Mice PMO-G was administered intravenously into adult mdx mice at 25 mg/kg/week for 3 weeks with additional glycine administration every other day intravenously followed by 25 mg/kg/month for 5 months with additional glycine administration every week intravenously. (A) Diagram of dosing regimen for the long-term study in mdx mice. i.v., intravenous injection. (B) Immunohistochemistry for dystrophin expression in body-wide muscles from mdx mice treated with PMO-S or PMO-G (scale bar, 100 μm). (C) Western blot for dystrophin expression in body-wide muscles from mdx mice treated with PMO-G or PMO-S. 2.5, 5, 10, and 25 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. TA, tibialis anterior; Q, quadriceps; G, gastrocnemius; T, triceps; A, abdominal muscle; D, diaphragm. (D) Quantitative analysis of dystrophin expression in body-wide muscles from treated mdx mice (n = 4; two-tailed t test). (E) Re-localization of DAPC components in treated mdx mice to assess dystrophin function and recovery of normal myoarchitecture (scale bar, 50 μm). The arrowheads point to identical myofibers. (F) Measurement of serum creatine kinase (CK) levels in treated mdx mice (n = 4; one-way ANOVA and post hoc Student-Newman-Keuls test). (G) Muscle function was assessed to determine the physical improvement with grip strength test (n = 4; one-way ANOVA and post hoc Student-Newman-Keuls test). (H) Measurement of muscle endurance with the running wheel test (n = 4; one-way ANOVA and post hoc Student-Newman-Keuls test). Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.

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.

Figure 4

Glycine Potentiates PMO Activity by Replenishing One-Carbon Unit Pool (A) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from mdx mice treated with PMO-G and bitopertin (BP) (scale bar, 100 μm). Glycine or the mixture of glycine and BP was administered into mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (B) Quantitative analysis for PAX7⁺ and PAX7⁺/Ki67⁺ MuSCs in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (C) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (D) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). PMO (2 μg) in saline and other solutions was injected into mdx TA muscles and muscles were harvested 2 weeks later. (E) Western blot and quantitative analysis for dystrophin expression in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). 2.5 and 5 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. (F) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from treated mdx mice (scale bar, 100 μm). Glycine, formate, tetrahydrofolate (THF), or the mixture of glycine with methotrexate (MTX) or THF was injected in mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (G) Quantitative analysis for PAX7⁺ and PAX7⁺/Ki67⁺ MuSCs in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (H) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (I) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (J) Western blot and quantitative analysis for dystrophin expression in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). 0.5, 5, and 10 μg of total protein from C57BL/6 mice and 50 μg from muscle samples from untreated and treated mdx mice were loaded. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.

Figure 5

Disruption of Glycine Decarboxylase (GLDC) Compromises the Functionality of Glycine in mdx Mice (A) Western blot analysis to determine GLDC knockdown efficiency at the protein level in TA muscles transfected with GLDC shRNA-expressing AAV2/8 viruses in mdx mice 3 weeks later (n = 3). SC refers to AAV2/8 expressing scramble shRNA. (B) Immunohistochemistry for PAX7⁺ MuSCs in TA muscles from treated mdx mice (scale bar, 100 μm). The arrowheads point to PAX7⁺ MuSCs. (C) Quantitative analysis for PAX7⁺ MuSCs in TA muscles from treated mdx mice (n = 3). (D) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (n = 3) (scale bar, 100μm). (E) Immunohistochemistry for dystrophin-positive fibers in TA muscles from treated mdx mice (scale bar, 100 μm). (F) Western blot and quantitative analysis for dystrophin expression in TA muscles from treated mdx mice (n = 4). 2.5 and 5 μg of total protein from C57BL/6 mice and 50 μg of muscle samples from untreated and treated mdx mice were loaded. Two-tailed t test was used for statistical analysis. ∗p < 0.05, ∗∗p < 0.001. Data are presented as means ± SEM.

Figure 6

Glycine Augments PMO Activities by Heightening mTORC1 Activation in mdx Mice (A) Western blot to detect phosphorylated mTOR, S6K1, and S6 expression in quadriceps from mdx mice treated intravenously with PMO-S or PMO-G at 25 mg/kg/week for 3 weeks with additional supply of glycine every other day for 5 weeks. α-Actinin was used as the loading control. 50 μg of total protein was loaded. (B) Quantitative analysis of the ratio of phosphorylated mTOR, S6K1, and S6 to mTOR, S6K1, and S6 total protein expression, respectively (n = 3; two-tailed t test). (C) Immunohistochemistry for PAX7⁺ and Ki67⁺ MuSCs in TA muscles from treated mdx mice (scale bar, 100 μm). Glycine (Gly) or the mixture of glycine with PP242 (Gly/PP242) was injected into mdx TA muscles and muscles were harvested 3 days later. The arrowheads point to PAX7⁺ and Ki67⁺ MuSCs. (D) Quantitative analysis for PAX7⁺ and PAX7⁺/Ki67⁺ MuSCs in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (E) Immunohistochemistry and quantitative analysis for eMyHC⁺ regenerating myofibers in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test) (scale bar, 100μm). (F) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from treated mdx mice (scale bar, 100 μm) (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). PMO (2 μg) in saline (PMO-S), glycine (PMO-G), or the mixture of glycine with PP242 (PMO-G/PP242) was injected into mdx TA muscles and muscles were harvested 2 weeks later. (G) Western blot and quantitative analysis for dystrophin expression in TA muscles from treated mdx mice (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). 2.5 and 5 μg of total protein from C57BL/6 mice and 50 μg of muscle samples from untreated and treated mdx mice were loaded. (H) Hierarchical clustering analysis of cell cycle-related gene expression profiles in primary myoblasts isolated from mdx mice and then treated with 0.8 mM glycine for 24 h. Expression levels (fold) are depicted in colors in which red represents upregulation and green means downregulation. (I) Quantitative real-time RT-PCR analysis of cell cycle-related gene expression in TA muscles from mdx mice treated with glycine every other day for 1 week intravenously (n = 3; two-tailed t test). CDK1, cyclin-dependent kinase 1; CDC20, cell division cycle 20; CCNB2, cyclin B2; CCNB1, cyclin B1; CCNA1, cyclin A1; CCNE1, cyclin E1. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.

Figure 7

Glycine Promotes mTORC1 Translocation via v-ATPase and RagB (A) Western blot to detect phosphorylated S6K1, S6, and 4EBP1 expression in starved C2C12 cells followed by re-stimulation of total amino acids and glycine. 30 μg of total protein was loaded and tubulin was used as a loading control. AA⁺ refers to the supplementation of total amino acids; Gly⁺ refers to the supplementation of glycine into starved cells; AA⁻ represents the depletion of total amino acids. (B) Quantitative analysis of the ratio of phosphorylated S6K1, S6, and 4EBP1 to total expression of counterparts (n = 3; one-way ANOVA and post hoc Student-Newman-Keuls test). (C and D) Immunocytochemistry for mTOR (C) and quantitative analysis of cells with mTOR at lysosomes (D) in starved C2C12 cells followed by re-stimulation of total amino acids or glycine (scale bar, 10 μm) (n = 10; one-way ANOVA and post hoc Student-Newman-Keuls test). LAMP1 and LAMP2 were used as lysosome markers. (E and F) Immunocytochemistry for mTOR (E) and quantitative analysis of cells with mTOR at lysosomes (F) in HEK293T cells treated with glycine and BAF (scale bar, 10 μm) (n = 10; two-tailed t test). (G) Western blot to detect phosphorylated S6K1 and S6 expression in HEK293T cells treated with glycine or glycine and BAF. 30 μg of total protein was loaded and tubulin was used as a loading control. (H) Western blot to detect phosphorylated S6K1 and S6 expression in RagB knockout HEK293T cells treated with glycine or total amino acids. 30 μg of total protein was loaded and GAPDH was used as a loading control. RagB CON refers to normal HEK293T cells; RagB KO means RagB knockout HEK293T cells. Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.

Figure 8

Effects of Glycine on Cell Transplantation in mdx Mice (A) Schematics for the cell transplantation in immunosuppressed mdx mice. i.v., intravenous injection; −7, 7 days prior to cell transplantation; −1, 1 day prior to cell transplantation. mdx mice were immunosuppressed during the experimental period unless otherwise specified. (B) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from glycine-treated mdx mice transplanted with wild-type MuSCs and muscles were harvested 3 weeks later (scale bar, 100 μm) (n = 4; two-tailed t test). (C) Measurement of TA muscle weight from mdx mice transplanted with MuSCs 3 weeks after transplantation. mdx refers to mdx controls without cell transplantation (n = 4). (D) Immunohistochemistry and quantitative analysis for dystrophin-positive fibers in TA muscles from glycine-treated mdx mice transplanted with wild-type primary myoblasts 3 weeks after transplantation (scale bar, 100 μm) (n = 4; two-tailed t test). (E) Tissue imaging to examine the GFP fluorescence and quantitative analysis of fluorescence intensity in glycine-treated mdx mice transplanted with wild-type GFP-positive MuSCs 3 weeks after transplantation (scale bar, 100 μm) (n = 6; one-way ANOVA and post hoc Student-Newman-Keuls test). (F and G) Immunohistochemistry (F) and quantitative analysis for dystrophin- and GFP-positive fibers (G) in TA muscles from glycine-treated mdx mice transplanted with GFP-positive MuSCs 3 weeks later (scale bar, 100 μm) (n = 6; two-tailed t test). Data are presented as means ± SEM. ∗p < 0.05, ∗∗p < 0.001.