miR-379 links glucocorticoid treatment with mitochondrial response in Duchenne muscular dystrophy

Abstract

Duchenne Muscular Dystrophy (DMD) is a lethal muscle disorder, caused by mutations in the DMD gene and affects approximately 1:5000-6000 male births. In this report, we identified dysregulation of members of the Dlk1-Dio3 miRNA cluster in muscle biopsies of the GRMD dog model. Of these, we selected miR-379 for a detailed investigation because its expression is high in the muscle, and is known to be responsive to glucocorticoid, a class of anti-inflammatory drugs commonly used in DMD patients. Bioinformatics analysis predicts that miR-379 targets EIF4G2, a translational factor, which is involved in the control of mitochondrial metabolic maturation. We confirmed in myoblasts that EIF4G2 is a direct target of miR-379, and identified the DAPIT mitochondrial protein as a translational target of EIF4G2. Knocking down DAPIT in skeletal myotubes resulted in reduced ATP synthesis and myogenic differentiation. We also demonstrated that this pathway is GC-responsive since treating mice with dexamethasone resulted in reduced muscle expression of miR-379 and increased expression of EIF4G2 and DAPIT. Furthermore, miR-379 seric level, which is also elevated in the plasma of DMD patients in comparison with age-matched controls, is reduced by GC treatment. Thus, this newly identified pathway may link GC treatment to a mitochondrial response in DMD.

Figure 1

GRMD cohort and miRNA expression. 1a. Phenotype variations through ages of GRMD dogs. 1b. Fold change of normalized mRNA abundance in muscle biopsies of 2-month old dogs (n = 9–10). CD11b: CD11 Antigen-Like Family Member, Col6a: collagen 6a3; MYH8: myosin heavy chain 8; BF: Biceps Femoris; SC: Sartorius Cranialis. 1c. Heat map classification of miRNA dysregulation in GRMD muscle biopsies, Biceps Femoris (BF) and Sartorius Cranialis (SC) moderate and severe phenotypes. The myomiRs and miRNAs from the Dlk1-Dio3 locus are marked by black arrows. 1d. Principal component analysis of muscle (combined SC and BF) miRNA expression in dog groups, wild type (WT), GRMD severe (SEV), and GRMD moderate (MOD). 1e. Principal component analysis of miRNA expression in GRMD muscles (combined moderate and severe group) Sartorius cranialis (SC) and biceps femoral (BF). Circles mark the aggregation of the individuals according to their group. Small circles denoted 95% confident interval of the gravity center, while the larger discontinued-line circles surrounds 95% of the dots.

Figure 2

miR 379 target validation, EIF4G2 and DAPIT regulation by miR-379. 2a. miR-379 expression in GRMD muscle biopsy (a graphical presentation of miR-379 data of Table 1, (n = 9 or 10)); BF Biceps femoris; SC Sartorius cranialis. (b) Bioinformatics prediction of the binding site of miR-379 on EIF4G2 in human, dog and mouse. MiR-379-5p seed and complementary targets sequences are in respectively in blue and red. (c) EIF4G2 expression in miR-379 and miR-139 transfected AB1190 human myoblasts, shown representative results of three experiments. (d) Graphical presentation of (c). (e) Schematic description of the IP-Ago method for miRNA target validation. (f) IP-Ago enrichment for miR-379 targets sequences in AB1190 human myoblasts, shown result of one out of two independent experiments. (g) and (h) EIF4G2 and DAPIT mRNA (2 g) and protein expressions (2 h) in miR-379 overexpressing AB1190 myoblasts. (i,j) EIF4G2 and DAPIT mRNA (2i) and protein expressions (2j) in shEIF4G2 overexpressing AB1190 myoblasts.

Figure 3

Myogenic expression of miR-379, EIF4G2 and DAPIT. 3a. Images of freshly isolated, proliferating and differentiated muscle stem cells (MuSC). Scale bar = 200 µm. (b) miR-379 relative abundance in freshly isolated Fr-MuSC; Day 4 proliferation MuSC (D4 prolif MuSC), day 2 and 5 of differentiation of the same cells. (c) to (e) EIF4G2 and DAPIT mRNA (c) and protein (d,e) expressions during AB1190 human myoblasts proliferation (day 0) and differentiation (Days 1, 2 and 3). (f) Correlation of DAPIT to EIF4G2 protein expression in AB1190 myoblasts during proliferation (Day 0) and differentiation (Days 1, 2 and 3). Dotted line = linear regression. (g) immunofluorescence detection of EIF4G2 and DAPIT in transversal sections of the quadriceps muscle of adult mdx and C57Bl/10 mouse.

Figure 4

Myogenic effect of inhibition of EIF4G2 and DAPIT. (a,b) Knocking down of EIF4G2 and DAPIT expression delays differentiation of AB1190 myoblast. AB1190 myoblast were treated by shRNA (shScramble, shEIF4G2, shDAPIT). Cells were differentiated for 4 days. Myotubes containing 6 or more nuclei (yellow arrows in representative images in 3 h) were counted (presented graphically in b). (c,d) Muscle regulatory factors (MRF) expression in AB1190 myoblast with knocked-down expression by shRNA of EIF4G2 (c) and DAPIT (d). (e,f) Transcriptional expression of genes associated with myogenic differentiation in shRNA knock-down EIF4G2 (e) and DAPIT (f).

Figure 5

DAPIT IHC localization, mitochondrial functional studies in M180 iPS cells. (a) Co-localization of DAPIT with ATP5a (mitochondrial complex 5) into oxidative fibers (NADH positive) in transversal section of quadriceps muscle from adult C57Bl/10 mouse. (b) Co-localization DAPIT and ATP5a in single fibers isolated from the gastrocnemius of adult C57Bl/10 mouse. (c,d) EIF4G2 and DAPIT mRNA (c) and protein expressions (d) in EIF4G2 and DAPIT knocked-down M180 iPS-derived skeletal myoblasts (skMPC). (e,f,g) Mitochondrial complex 5 to 1 activity ratio (e) and absolute activity (f,g) in EIF4G2 and DAPIT knocked-down skMPC. (h,i) TFAM (h) and PGC-1α (i) expression in knocked-down skMPC. (j) ATP to ADP ration in DAPIT knockdown (sh-DAPIT) versus control (sh-Scramble) and untreated AB1190 (n = 4, in 3 independent experiments) in vitro differentiated myotubes.

Figure 6

Glucocorticoid response of miR-379, EIF4G2 and DAPIT. (a) Foxo1, EIF4G2 and USMG5 mRNAs (normalized to p0 (RPLP0)), and miR-379-5p (normalized to U6/miR-93/miR-16), relative abundance in the gastrocnemius muscle of C57Bl/10 (n = 11), treated by dexamethasone (D), and untreated control (C). (b) and (c), Protein expression levels of Foxo1, EIF4G2 and DAPIT (normalized to total protein) in the gastrocnemius muscle of C57Bl/10 treated by dexamethasone (D) and untreated (C). (d) miR-139-5p and miR-379-5p expression in the plasma in a human cohort (each dot represents one patient, n = 9). Healthy control (Cont), DMD patients, untreated (DMD UT), and treated by glucocorticoid (DMD GC), of the age groups 4–8, 8–12 and 12–20 years old.

Figure 7

Revised model for mitochondria dysfunction in the DMD disease. Graphical model for mechanisms of mitochondrial dysfunction in the DMD disease. The Ca²⁺ hypothesis, which is presented on the model upper part, was proposed initially by Wrogemann and Pena at 1976. Accordingly, in the absence of dystrophin, Ca²⁺ entry via sarcolemma leakiness and/or activated ion channels is initiating a cytoplasmic pathological cascade (see main text for details) that culminate by the opening of the mitochondrial permeability transition pore (mPTP) and mitochondrial degeneration. In the modified model, presented on the image lower part, it is proposed that the absence of dystrophin induces gene-expression changes, activating miR-379 expression, reducing EIF4G2 and DAPIT expression, reducing ATP synthase activity and ATP production, (see main text for details), which might synergizes with the external Ca²⁺ overload, to produce mitochondrial damage.