Cholesterol metabolism is a potential therapeutic target in Duchenne muscular dystrophy

Abstract

Background: Duchenne muscular dystrophy (DMD) is a lethal muscle disease detected in approximately 1:5000 male births. DMD is caused by mutations in the DMD gene, encoding a critical protein that links the cytoskeleton and the extracellular matrix in skeletal and cardiac muscles. The primary consequence of the disrupted link between the extracellular matrix and the myofibre actin cytoskeleton is thought to involve sarcolemma destabilization, perturbation of Ca²⁺ homeostasis, activation of proteases, mitochondrial damage, and tissue degeneration. A recently emphasized secondary aspect of the dystrophic process is a progressive metabolic change of the dystrophic tissue; however, the mechanism and nature of the metabolic dysregulation are yet poorly understood. In this study, we characterized a molecular mechanism of metabolic perturbation in DMD.

Methods: We sequenced plasma miRNA in a DMD cohort, comprising 54 DMD patients treated or not by glucocorticoid, compared with 27 healthy controls, in three groups of the ages of 4-8, 8-12, and 12-20 years. We developed an original approach for the biological interpretation of miRNA dysregulation and produced a novel hypothesis concerning metabolic perturbation in DMD. We used the mdx mouse model for DMD for the investigation of this hypothesis.

Results: We identified 96 dysregulated miRNAs (adjusted P-value <0.1), of which 74 were up-regulated and 22 were down-regulated in DMD. We confirmed the dysregulation in DMD of Dystro-miRs, Cardio-miRs, and a large number of the DLK1-DIO3 miRNAs. We also identified numerous dysregulated miRNAs yet unreported in DMD. Bioinformatics analysis of both target and host genes for dysregulated miRNAs predicted that lipid metabolism might be a critical metabolic perturbation in DMD. Investigation of skeletal muscles of the mdx mouse uncovered dysregulation of transcription factors of cholesterol and fatty acid metabolism (SREBP-1 and SREBP-2), perturbation of the mevalonate pathway, and the accumulation of cholesterol in the dystrophic muscles. Elevated cholesterol level was also found in muscle biopsies of DMD patients. Treatment of mdx mice with Simvastatin, a cholesterol-reducing agent, normalized these perturbations and partially restored the dystrophic parameters.

Conclusions: This investigation supports that cholesterol metabolism and the mevalonate pathway are potential therapeutic targets in DMD.

Keywords: Biological interpretation of miRNA dysregulation; Cholesterol; DLK1-DIO3; Duchenne muscular dystrophy; Glucocorticoid; Host gene; Lipid metabolism; SREBP-1; SREBP-2; Simvastatin.

Figure 1

DMD cohort characterization. (A) cohort subgroups and age composition. DMD patients and healthy controls were classified into three age groups of 4–8, 8–12 and, 12–20 years old. DMD patients were glucocorticoid treated (DMD T) or untreated (DMD UT). The figurine symbols represent the ages of individual patients on the horizontal axis, black for untreated DMD, grey figures for treated DMD, and brown figures for healthy controls. Treated and untreated DMD of the 4–8 group of age are the same patients before and after GC treatment (except one patient). (B) A graphical presentation of the spectrum of dystrophin mutations by age group. Dystrophin's gene 79 exons and protein domains are presented on respectively the upper and lower vertical bands. Patients of the 4–8 age years old group are represented twice in the cohort (with the exceptions of D8 and D17), before and after glucocorticoid treatment, by samples D1 to D9 and D10 to D18, respectively. Del (blue) = deletion; dup (red) = duplication; St (black) = stop codon mutation; poi (in black) = point mutation.

Figure 2

Characterization of circulating RNA by high throughput sequencing. (A) schematic presentation of plasma sample processing. Small RNA high quality reads (high quality reads) were mapped on the human genome (mapped reads). Mapped reads were classified to miRNA and other small RNA classes. (B) Graphical presentation of high quality reads (blue), mapped reads (red), and miRNA (green) in the cohort subgroups are presented as average ± SEM. (C) PCA analysis of cohort segregation according to miRNA expression in DMD (orange dots) and healthy control (green dots) by age groups (panels 1 to 5) and treated (red dots) versus untreated (black dots) DMD patients (panel bottom right).

Figure 3

Plasma miRNA profiling in the DMD cohort. (A) a volcano plot of miRNA dysregulation in the plasma of 4–12 years old DMD patients (treated and untreated together, n = 36) versus healthy control (n = 18). Up‐regulated miRNA in DMD are on the right side and down‐regulated on the left side of the threshold lines (|FC| ≥ 1.5). MiRNAs above the horizontal line are differentially expressed with adjusted p < 0.1). DystromiRs in yellow, cardiomiRs in red, DLK1‐DIO3 miRs in green, Let‐7 family miR in blue, miR‐320 family in brown, unclassified miR in black. (B) A graphical presentation of miRNA up‐regulated in DMD. (C) A graphical presentation of miRNA down‐regulated in DMD. (D) Glucocorticoid responsive miRNAs in DMD. Each dot represents one patient, n = 9, error bar = SEM.

Figure 4

Bioinformatic analysis of host‐genes for dysregulated miRNAs in DMD plasma. (A) Network 1: Lipid metabolism, molecular transport, small molecule biochemistry, neurological disease, cancer, organismal, injury and abnormalities. (B) Network 2: lipid metabolism, small molecule biochemistry, molecular transport. (C) Network 3, lipid metabolism, small molecule biochemistry, dermatological diseases and conditions. (D) Merged networks 1–3, lipid metabolism. The connected molecules in each network are in blue. Red is up‐regulated, green is down‐regulated, continuous and discontinuous arrows are respectively direct and indirect relations, except in (D). (E) GO terms of host genes for dysregulated miRNAs, classified by P‐value. (F) Lipid dysregulation network in DMD. A graphical presentation of a sub‐selection of miRNA host genes, which are participating in lipid metabolism, and their related GO terms. Round circle = complex; rhombus = enzyme; inverse triangle = kinase; flat (horizontally oriented) circle = transcription regulator; vertically oriented circle = transmembrane receptor; trapeze = transporter.

Figure 5

SREBP pathway in the mdx muscle. (A) A schematic presentation of the components in the SREBP pathway (see text for details). (B, C) Fold change SREBP pathway transcripts in the gastrocnemius (B) and the diaphragm (C) in the mdx versus healthy control mouse. (D–G) A western blot analysis of SREBP pathway protein expression in the gastrocnemius (D) and its graphical quantification (E), and of the diaphragm muscle (F) and its graphical quantification (G). SREBP‐1 = sterol regulatory binding element 1; SREBP‐2 = sterol binding regulatory element 2; HMGCR = HMG‐CoA reductase; FASN = fatty acid synthase; LDLR = low density lipoprotein receptor; SCAP=SREBP cleavage‐activating protein.

Figure 6

Simvastatin effect on skeletal muscle in the mdx mouse. Seven‐week‐old (young adult) control mice, and mdx mice untreated (mdx) or treated (mdx‐Simva) by simvastatin during a 3 weeks period (n = 6). (A, B) Serum myomesin‐3 (Myom3) and its graphical presentation. (C) Muscle creatine kinase (mCK) in the blood serum. (D, E) fibrosis staining (Sirius red) of diaphragm transversal sections and its graphical presentation. (F) Images of free cholesterol staining (Filipin) of transversal sections of a whole diaphragm. (G, H) Confocal images of diaphragm transversal sections (G) and its quantification (H). (I) Cholesterol content of skeletal muscle biopsies of DMD patients and their healthy controls (n = 4). (J, K) A western blot analysis (J) and its graphical presentation (K) of SREBP‐1, SREBP‐2 and HMGCR in the diaphragm muscle of control, mdx, and treated mdx mice. (L, M) Confocal microscopy images of SREBP‐1, SREBP‐2 in the diaphragms of the same mice. Notice that the wheat germ agglutinin (WGA) lectin stains both the sarcolemma and the myonuclear membranes. Blue arrows denote SREBP positive myonucleus inside regenerated myofibres (in the centre of the myofibre). Pink arrows denote SREBP negative myonuclei in regenerated myofibres.