Glycine administration attenuates progression of dystrophic pathology in prednisolone-treated dystrophin/utrophin null mice

Abstract

Duchenne muscular dystrophy (DMD) is an X-linked genetic disease characterized by progressive muscle wasting and weakness and premature death. Glucocorticoids (e.g. prednisolone) remain the only drugs with a favorable impact on DMD patients, but not without side effects. We have demonstrated that glycine preserves muscle in various wasting models. Since glycine effectively suppresses the activity of pro-inflammatory macrophages, we investigated the potential of glycine treatment to ameliorate the dystrophic pathology. Dystrophic mdx and dystrophin-utrophin null (dko) mice were treated with glycine or L-alanine (amino acid control) for up to 15 weeks and voluntary running distance (a quality of life marker and strong correlate of lifespan in dko mice) and muscle morphology were assessed. Glycine increased voluntary running distance in mdx mice by 90% (P < 0.05) after 2 weeks and by 60% (P < 0.01) in dko mice co-treated with prednisolone over an 8 week treatment period. Glycine treatment attenuated fibrotic deposition in the diaphragm by 28% (P < 0.05) after 10 weeks in mdx mice and by 22% (P < 0.02) after 14 weeks in dko mice. Glycine treatment augmented the prednisolone-induced reduction in fibrosis in diaphragm muscles of dko mice (23%, P < 0.05) after 8 weeks. Our findings provide strong evidence that glycine supplementation may be a safe, simple and effective adjuvant for improving the efficacy of prednisolone treatment and improving the quality of life for DMD patients.

Figure 1

Glycine improves muscle function in mdx dystrophic mice. Dose-response effect of glycine feeding on running performance in mdx mice (A), with no effect of L-alanine (control). Three weeks of glycine feeding improved whole-body function (B–D), tended to improve TA peak tetanic force (F) but not muscle mass-normalized peak tetanic force (G) or diaphragm specific force (E), TA fatigue (H), body mass (I) or muscle mass normalized to body mass and BL/10 (J). The data are mean ± SD of 10–12 animals per group. Data were analyzed using one-way ANOVAs with Fishers LSD or Tukey’s (A) post hoc test, except for (D) which was analyzed using a log-rank Mantel-Cox test. *denotes p < 0.05; **denotes p < 0.01; ***denotes p < 0.001.

Figure 2

Long-term glycine feeding improves voluntary running and reduces fibrosis in mdx dystrophic mice. The effect of glycine or L-alanine feeding in mdx mice on mRNA expression of inflammatory mediators (A, interleukin-6, Il-6; suppressor of cytokine signaling 3, Socs3; EGF-like module-containing mucin-like hormone receptor-like 1, F4/80; chemokine ligand 2, Ccl2; mannose receptor, Cd206) and factors involved in fibrosis/collagen synthesis (B), protein disulphide-isomerase, P4hb; transforming growth factor beta, Tgfβ; collagen type I, III and IV, Col1a1, Col3a1 and Col4a1 and; matrix metallopeptidase 2, Mmp2). The PCR data are normalized to the BL/10 controls and presented as mean ± SD of 10–12 animals per group. The effect of chronic glycine feeding on running performance in mdx mice (C) compared to L-alanine (control). Representative muscle cross-sections stained with Van Gieson’s to visualize collagen and its quantification from mdx mice after chronic treatment with glycine (D). Scale bar represents 100 µm. Data were analyzed using one-way ANOVAs with Fishers LSD post hoc test. *denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001.

Figure 3

Glycine blunts diaphragm fibrosis but is insufficient as a standalone treatment to extend lifespan in severely dystrophic dko mice. The effect of glycine or L-alanine feeding in dko mice on body mass (A), TA (B) and diaphragm (C) muscle function, TA mass (D) and voluntary running distance (E). Representative muscle cross-sections stained with Van Gieson’s to visualize collagen and its quantification from dko mice after chronic treatment with glycine (F). Scale bar represents 100 µm. Voluntary running distance in mdx, mdx:utrophin^+/− and dko treated with either glycine or L-alanine for 2 weeks (G). Survival curve of L-alanine and glycine-treated dko mice (H); changes in running distance and body mass throughout the lifespan of dko mice (I); and correlation (Pearson’s r) between lifespan and the natural logarithm of running distance (J). Data in A-E were analyzed using two-way ANOVAs with Sidak’s post hoc test. Two-group comparisons were made using students t-tests, which lifespan curves were compared using a log-rank Mantel-Cox test. Data are mean ± SD of at least 8 mice per group.

Figure 4

Glycine treatment augments prednisolone-induced improvements in voluntary wheel running and diaphragm fibrosis in dko mice. Endpoint body mass (A), absolute muscle mass normalized to BL/10 (B) muscle mass normalized to body mass and BL/10 (C), as well as weekly (D) and average (E) voluntary running distance in dko mice receiving prednisolone with either glycine or L-alanine for 8 weeks. Individual muscle mass in dko mice after 8 weeks of treatment (C). Assessments of whole body function including forelimb grip strength (F) and inverted hang time (G). The effect of glycine and prednisolone treatment on in situ peak tetanic force of the TA muscle at different stimulation frequencies (H), ex vivo peak specific force of diaphragm muscle in dko mice (I) and inflammatory gene expression (J). Representative muscle cross-sections stained with Van Gieson’s to visualize collagen and its quantification from dko (K) mice after 8 weeks of prednisolone and glycine treatment. Scale bar represents 100 µm. Data were analyzed using one-way ANOVAs with Fishers LSD post hoc test. * denotes p < 0.05; ** denotes p < 0.01; *** denotes p < 0.001. Data are presented as mean ± SD for 12–15 mice per group.