Lack of miR-378 attenuates muscular dystrophy in mdx mice
Paulina Podkalicka, Olga Mucha, Iwona Bronisz-Budzyńska, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Anna Cetnarowska, Urszula Głowniak-Kwitek, Karolina Bukowska-Strakova, Maciej Cieśla, Maria Kulecka, Jerzy Ostrowski, Michał Mikuła, Anna Potulska-Chromik, Anna Kostera-Pruszczyk, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak
Paulina Podkalicka, Olga Mucha, Iwona Bronisz-Budzyńska, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Anna Cetnarowska, Urszula Głowniak-Kwitek, Karolina Bukowska-Strakova, Maciej Cieśla, Maria Kulecka, Jerzy Ostrowski, Michał Mikuła, Anna Potulska-Chromik, Anna Kostera-Pruszczyk, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak
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Research Article Muscle biology

Lack of miR-378 attenuates muscular dystrophy in mdx mice

Abstract

The severity of Duchenne muscular dystrophy (DMD), an incurable disease caused by the lack of dystrophin, might be modulated by different factors, including miRNAs. Among them, miR-378 is considered of high importance for muscle biology, but intriguingly, its role in DMD and its murine model (mdx mice) has not been thoroughly addressed so far. Here, we demonstrate that dystrophic mice additionally globally lacking miR-378 (double-KO [dKO] animals) exhibited better physical performance and improved absolute muscle force compared with mdx mice. Accordingly, markers of muscle damage in serum were significantly decreased in dKO mice, accompanied by diminished inflammation, fibrosis, and reduced abundance of regenerating fibers within muscles. The lack of miR-378 also normalized the aggravated fusion of dystrophin-deficient muscle satellite cells (mSCs). RNA sequencing of gastrocnemius muscle transcriptome revealed fibroblast growth factor 1 (Fgf1) as one of the most significantly downregulated genes in mice devoid of miR-378, indicating FGF1 as one of the mediators of changes driven by the lack of miR-378. In conclusion, we suggest that targeting miR-378 has the potential to ameliorate DMD pathology.

Authors

Paulina Podkalicka, Olga Mucha, Iwona Bronisz-Budzyńska, Magdalena Kozakowska, Katarzyna Pietraszek-Gremplewicz, Anna Cetnarowska, Urszula Głowniak-Kwitek, Karolina Bukowska-Strakova, Maciej Cieśla, Maria Kulecka, Jerzy Ostrowski, Michał Mikuła, Anna Potulska-Chromik, Anna Kostera-Pruszczyk, Alicja Józkowicz, Agnieszka Łoboda, Józef Dulak

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Figure 2

Muscle damage markers in serum and inflammation in gastrocnemius muscle are decreased in 3-month-old dystrophic mice lacking miR-378.

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Muscle damage markers in serum and inflammation in gastrocnemius muscle ...
(A) Lower serum activity of LDH in mdx animals lacking miR-378; activity assay; n = 10–14/group. (B) Increased serum CK activity in mdx mice with a tendency to be decreased by miR-378-KO, activity assay; n = 11–13/group. (C) Necrosis assessment by immunofluorescent staining of IgM and IgG (green) binding and its calculation indicating no differences between groups; n = 9–10/group. Scale bar: 100 μm. (D) Representative pictures of H&E staining of gastrocnemius muscle with semiquantitative analysis of inflammation extent showing a tendency in decreased inflammatory cell infiltration in dKO mice; microscopic assessment using Nikon Eclipse microscope. Scale bar: 100 μm; n = 4–6/group. (E) Decreased number of WBC in the peripheral blood in dKO mice; blood cell count; n = 5–6/group. (F–J) The analysis of inflammatory cells in hind limb muscles with special emphasis on macrophage subpopulations; flow cytometry analysis calculated as the percentage of Hoechst+ cells; n = 5/group. The percentage of CD45+ cells (F), macrophages (CD45+F4/80+CD11b+ cells) (G), M1-like macrophages (CD45+F4/80+CD11b+MHCIIhiCD206lo cells) (H), M2-like macrophages (CD45+F4/80+CD11b+MHCIIloCD206hi cells) (I), and eosinophils (CD45+F4/80+CD86+ cells) (J) showing significant decrease in dKO mice. (K) The decreased HO-1 protein level in dKO animals assessed by Western blot; GAPDH used as loading control. Representative result of 2 independent experiments; n = 4–5/group. Data are presented as mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; (A–B and D–J) 1-way ANOVA with Tukey’s post hoc test; (C) unpaired 2-tailed Student’s t test.