Duchenne's muscular dystrophy involves a defective transsulfuration pathway activity

Abstract

Duchenne muscular dystrophy (DMD) is the most frequent X chromosome-linked disease caused by mutations in the gene encoding for dystrophin, leading to progressive and unstoppable degeneration of skeletal muscle tissues. Despite recent advances in the understanding of the molecular processes involved in the pathogenesis of DMD, there is still no cure. In this study, we aim at investigating the potential involvement of the transsulfuration pathway (TSP), and its by-end product namely hydrogen sulfide (H₂S), in primary human myoblasts isolated from DMD donors and skeletal muscles of dystrophic (mdx) mice. In myoblasts of DMD donors, we demonstrate that the expression of key genes regulating the H₂S production and TSP activity, including cystathionine γ lyase (CSE), cystathionine beta-synthase (CBS), 3 mercaptopyruvate sulfurtransferase (3-MST), cysteine dioxygenase (CDO), cysteine sulfonic acid decarboxylase (CSAD), glutathione synthase (GS) and γ -glutamylcysteine synthetase (γ-GCS) is reduced. Starting from these findings, using Nuclear Magnetic Resonance (NMR) and quantitative Polymerase Chain Reaction (qPCR) we show that the levels of TSP-related metabolites such as methionine, glycine, glutathione, glutamate and taurine, as well as the expression levels of the aforementioned TSP related genes, are significantly reduced in skeletal muscles of mdx mice compared to healthy controls, at both an early (7 weeks) and overt (17 weeks) stage of the disease. Importantly, the treatment with sodium hydrosulfide (NaHS), a commonly used H₂S donor, fully recovers the impaired locomotor activity in both 7 and 17 old mdx mice. This is an effect attributable to the reduced expression of pro-inflammatory markers and restoration of autophagy in skeletal muscle tissues. In conclusion, our study uncovers a defective TSP pathway activity in DMD and highlights the role of H₂S-donors for novel and safe adjuvant therapy to treat symptoms of DMD.

Keywords: Autophagy; Duchenne muscular dystrophy; H(2)S donors; Inflammation; Sodium hydrosulfide (NaHS).

Fig. 1

Scheme of transsulfuration pathway (TSP). In mammalians, the amino acid methionine could be converted into homocysteine. Homocysteine could be transformed, through two steps enzymatic reaction, into l-cysteine. l-cysteine acts as a substrate leading to the biosynthesis of three major final products: H₂S, taurine and glutathione. Abbreviation: CAT, cysteine aminotransferase; CBS, cystathionine beta-synthase; CDO cysteine dioxygenase; CSAD, cysteine sulfonic acid decarboxylase; CSE, cystathionine-γ-lyase; GS: glutathione synthase; HDD, hypotaurine dehydrogenase; MPST, 3-mercaptopyruvate sulfurtransferase; γ-GCS: Gamma-glutamylcysteine synthetase.

Fig. 2

mRNA expression levels of TSP genes in human primary myoblasts isolated from DMD donors. Bar graphs showing the CBS, CSE, 3-MST, CDO, CSAD, γ-GCS and GS mRNA expression levels in primary human myoblasts isolated from two healthy (HD1 and HD2) and four DMD donors (D1–D4). The quantification of transcripts has been performed in duplicate for each donor as indicated by the single dots shown on the graph. The difference in the expression levels of TSP genes are calculated by combining healthy and/or DMD donors together. Each bar is, therefore, the mean ± SEM of two healthy vs four DMD donors. *p ≤ 0.05 and **p ≤ 0.01 vs healthy donors.

Fig. 3

Levels of main metabolites regulating the transsulfuration pathway in skeletal muscles of 7 weeks old control and mdx mice measured by NMR. (A) Scatter plots displaying the metabolite distribution of all classes of metabolites identified in quadriceps femoris muscle of control and mdx mice. (B–F) Left, graphs showing the levels of methionine, glycine, glutamate, glutathione and taurine detected in each control wild type (wt) (n = 5) or dystrophic mouse (n = 6). Right, the bar graphs show the mean values ± SEM per group. Differences are considered statistically significant when p was ≤0.05. Single asterisk (*) or double asterisk (**) denote a p-value of ≤0.05 and 0.01 vs wt mice.

Fig. 4

Levels of main metabolites regulating the transsulfuration pathway in skeletal muscles of 17 weeks old control and mdx mice measured by NMR. (A) Scatter plots displaying the metabolite distribution of all classes of metabolites identified in quadriceps femoris muscle of control and mdx mice. (B–F) Left, graphs showing the levels of methionine, glycine, glutamate, glutathione and taurine detected in each control wt (n = 6) or dystrophic mouse (n = 6). Right, the bar graphs show the mean values ± SEM per group. Differences are considered statistically significant when p was ≤0.05. Double asterisk (**) and triple asterisk (***) denote a p-value of ≤0.01 and 0.001 vs wt mice.

Fig. 5

mRNA expression levels of TSP genes in skeletal muscles of mdx mice. Bar graphs showing the quantification of transcripts levels of CBS, CSE, 3-MST, CDO, CSAD, γ-GCS and GS evaluated by qPCR in QFA and gastrocnemius of control wt (n = 6) and mdx mice (n = 6). Control and mdx mice have been compared at both 7 (A–B) and 17 (C–D) weeks. Data are expressed as 2^∧-ΔΔct relative to ribosomal protein S16, as described in materials and methods. Differences are considered statistically significant when p was ≤0.05. Single asterisk (*), double-asterisk (**) and triple asterisk (***) denote a p-value of ≤0.05, 0.01 and 0.001 vs wt mice.

Fig. 6

Measurement of locomotor activity and histological analysis in control and mdx mice treated with NaHS. Muscle coordination and strength were evaluated by the weight (A) and rotarod (B) tests, measured in wt (n = 6) and mdx (n = 6) mice treated with vehicle or NaHS (3 mg kg⁻¹). Wt and mdx mice were daily treated (for two weeks), starting from week 5 to week 7 or up to week 17. Differences are considered statistically significant when p was ≤0.05. Double asterisk (**) and triple asterisk (***) denote a p-value of ≤0.05, 0.01 and 0.001 vs wt mice. Single circle (^○) and double circle (^○○) denote a p-value of ≤0.05 and 0.01 vs mdx mice of the same age. Histological analysis (C–H) has been performed on QFA of wt and mdx mice treated with vehicle or NaHS at 7 weeks (400× magnification). Typical histological hallmarks of dystrophic muscle (disorganized tissue architecture, cell infiltrates and fibrotic area) are highlighted in mdx mice by using H&E staining (D) and Goldner's trichrome staining (G). NaHS exerts a beneficial action on the amount of both cell infiltrate (see black arrow in panels D and E) and fibrotic area (see black arrow in G and H).

Fig. 7

mRNA expression levels of inflammation markers in skeletal muscles of mdx mice treated with NaHS. Bar graphs showing the mRNA expression levels of tumour necrosis factor-alpha (TNFα), interleukin 6 (IL-6), interleukin 1β (IL-1β) and transforming growth factor-beta (TGF-β) in quadriceps femoris of control and mdx mice receiving or not NaHS of both 7 (A–D) and 17 (E–D) weeks. Data are expressed as 2^∧-ΔΔct relative to ribosomal protein S16, as described in materials and methods. Single asterisk (*) and double asterisk (**) denote a p-value of ≤0.05 and 0.01 vs wt mice of the same age. Single circle (^○) and double circle (^○○) denote a p-value of ≤0.05 and 0.01 vs mdx group of the same age.

Fig. 8

mRNA expression levels of autophagy markers in skeletal muscles of mdx mice treated with NaHS. (A) Heatmap representation of selected genes obtained from PCR array analysis performed in QFA muscles isolated from 7-week-old control (n = 6) and mdx mice treated with vehicle (n = 6) or NaHS (n = 6). Red, physiological gene expression; green, gene downregulation. (B–E) Bar graphs showing the mRNA expression levels of Atg3, Atg7, Atg12 and Ulk1 in quadriceps femoris of 7 weeks old control and mdx mice receiving or not NaHS. Data are expressed as 2^∧-ΔΔct relative to actin, as described in materials and methods. (F–I) Western blot analysis showing the changes in the expression and/or phosphorylation of the two markers of autophagy/apoptosis AKT and LC3 in QFA of control and mdx mice treated or not with NaHS. Fold data represent the mean ± SEM of three separate experiments. The blots shown are representative of three independent experiments with similar outcomes. The GAPDH bands confirm that similar amounts of proteins were loaded on the gel for each sample. Single asterisk (*), double asterisk (**) and triple asterisk (***) denote a p-value of ≤0.05, 0.01, 0.001 vs wt mice, respectively. Single circle (^○), double circle (^○○) and triple circle (^○○○) denote a p-value of ≤0.05, 0.01, 0.001 vs mdx group, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 9

Graphical abstract.