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. 2022 Oct 10;7(19):e159875.
doi: 10.1172/jci.insight.159875.

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy

Zachary M Howard  1 , Chetan K Gomatam  1 , Charles P Rabolli  1   2 , Jeovanna Lowe  1 , Arden B Piepho  1 , Shyam S Bansal  1   2 , Federica Accornero  1   2 , Jill A Rafael-Fortney  1
Affiliations

Mineralocorticoid receptor antagonists and glucocorticoids differentially affect skeletal muscle inflammation and pathology in muscular dystrophy

Zachary M Howard et al. JCI Insight. .

Abstract

Mineralocorticoid receptor antagonists (MRAs) slow cardiomyopathy in patients with Duchenne muscular dystrophy (DMD) and improve skeletal muscle pathology and function in dystrophic mice. However, glucocorticoids, known antiinflammatory drugs, remain a standard of care for DMD, despite substantial side effects. Exact mechanisms underlying mineralocorticoid receptor (MR) signaling contribution to dystrophy are unknown. Whether MRAs affect inflammation in dystrophic muscles and how they compare with glucocorticoids is unclear. The MRA spironolactone and glucocorticoid prednisolone were each administered for 1 week to dystrophic mdx mice during peak skeletal muscle necrosis to compare effects on inflammation. Both drugs reduced cytokine levels in mdx quadriceps, but prednisolone elevated diaphragm cytokines. Spironolactone did not alter myeloid populations in mdx quadriceps or diaphragms, but prednisolone increased F4/80hi macrophages. Both spironolactone and prednisolone reduced inflammatory gene expression in myeloid cells sorted from mdx quadriceps, while prednisolone additionally perturbed cell cycle genes. Spironolactone also repressed myeloid expression of the gene encoding fibronectin, while prednisolone increased its expression. Overall, spironolactone exhibits antiinflammatory properties without altering leukocyte distribution within skeletal muscles, while prednisolone suppresses quadriceps cytokines but increases diaphragm cytokines and pathology. Antiinflammatory properties of MRAs and different limb and respiratory muscle responses to glucocorticoids should be considered when optimizing treatments for patients with DMD.

Keywords: Cytokines; Innate immunity; Muscle Biology; Skeletal muscle.

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Conflict of interest statement

Conflict of interest: The authors have declared that no conflict of interest exists.

Figures

Figure 1
Figure 1. Cytokine and chemokine levels in spironolactone- and prednisolone-treated mdx skeletal muscles.
(A) Proteome profiler cytokine array immunoblots incubated with lysates from spironolactone-treated (SPR-treated) and prednisolone-treated (PRD-treated) 5.5-week-old mdx quadriceps compared with vehicle-treated (VEH-treated) controls (5 mg of protein pooled from n = 5 SPR, n = 4 PRD, n = 5 VEH). (B) Immunoblots incubated with lysates from SPR- and PRD-treated 5.5-week-old mdx diaphragms compared with VEH-treated controls (5 mg of protein). (C and D) Immunoblot pixel densitometry bar graph displayed as a ratio comparing cytokine and chemokine levels between SPR- and VEH-treated mdx quadriceps and mdx diaphragms. (E and F) Immunoblot pixel densitometry bar graph displayed as a ratio comparing cytokine and chemokine levels between PRD- and VEH-treated mdx quadriceps and mdx diaphragms. A trend-line (dashes) is placed at y = 1 on each bar graph to visualize upregulated and downregulated cytokines and chemokines. (G) ELISA for IL-1β on soluble protein extracts from quadriceps muscles isolated from mdx mice treated for 2 weeks with VEH (n = 5), SPR (n = 5), or PRD (n = 4) compared with untreated WT control (C57) (n = 3). Statistics used were ANOVA with Dunnett’s test comparing each group with the VEH. *P ≤ 0.05.
Figure 2
Figure 2. Muscle leukocyte analysis in spironolactone- and prednisolone-treated mdx quadriceps.
(A–F) Representative flow cytometry gating dot plots displaying CD45+CD11b+ myeloid cells (A), CD45+CD11b+LY6G+ neutrophils (B), CD45+CD11b+LY6G–CD64–LY6Chi infiltrating monocytes (C), CD45+CD11b+LY6G–CD64+ macrophages (D), CD45+CD11b+LY6G–CD64+CD206+ macrophages (E), and CD45+CD11b+LY6G–CD64+F4/80hi macrophages (F) from spironolactone-treated (SPR-treated) (n = 13) and prednisolone-treated (PRD-treated) (n = 13) 4.5-week-old mdx quadriceps compared with vehicle (VEH) (n = 12) controls. (G) Quantification of myeloid cells, neutrophils (Nφ), infiltrating monocytes (Inf. MO), macrophages (MΦ), CD206+ macrophages (CD206+), and F4/80hi macrophages represented as dot plots for cells per milligram of muscle (Cells/mg). (H) Bar graphs with individual data points as percentages of total CD45+ leukocytes (%CD45+), CD45+ CD11b+ myeloid cells (%CD45+CD11b+) or macrophages (%CD45+CD11b+LY6G–CD64+) comparing SPR- and PRD-treated mice with VEH controls. Experimental replicates are denoted by black and gray dots within the graphs. Statistics used were ANOVA with the BKY test *P ≤ 0.05.
Figure 3
Figure 3. Muscle leukocyte analysis in spironolactone- and prednisolone-treated mdx diaphragms.
(A–F) Representative flow cytometry gating dot plots displaying CD45+CD11b+ myeloid cells (A), CD45+CD11b+LY6G+ neutrophils (B), CD45+CD11b+LY6G–CD64–LY6Chi infiltrating monocytes (C), CD45+CD11b+LY6G–CD64+ macrophages (D), CD45+CD11b+LY6G–CD64+CD206+ macrophages (E), and CD45+CD11b+LY6G–CD64+F4/80hi macrophages (F) from spironolactone-treated (SPR-treated) and prednisolone-treated (PRD-treated) 4.5-week-old mdx diaphragms compared with vehicle (VEH) controls. n = 6 replicates per group pooled from 2 mice each were used. (G) Quantification of myeloid cells, neutrophils (Nφ), infiltrating monocytes (Inf. MO), macrophages (MΦ), CD206+ macrophages (CD206+), and F4/80hi macrophages represented as dot plots for cells per milligram of muscle (Cells/mg). (H) Bar graphs with individual data points as percentages of total CD45+ leukocytes (%CD45+), CD45+CD11b+ myeloid cells (%CD45+CD11b+), or macrophages (%CD45+CD11b+LY6G–CD64+) comparing SPR- and PRD-treated mice with VEH controls. Experimental replicates are denoted by black and gray dots within the graphs. Statistics used were ANOVA with the BKY test *P ≤ 0.05.
Figure 4
Figure 4. Principal component analysis (PCA), overview of differentially expressed genes, and biological process GO analysis of sequenced RNA from spironolactone- and prednisolone-treated mdx quadriceps myeloid cells.
(A) Principal component analysis revealed 3 distinct groupings based on treatment condition. n = 3 replicates pooled from 3 mice each were used for each group. (B) Heatmap of all 896 genes that are differentially expressed between either prednisolone treatment versus vehicle, or spironolactone treatment versus vehicle. (C) Overlap analysis reveals 53 genes that are differentially expressed in both prednisolone versus vehicle and spironolactone versus vehicle. Prednisolone and spironolactone treatments yield 633 and 210 gene, respectively, that are uniquely differentially expressed in those treatment conditions versus vehicle. (D) GO analysis for biological processes.
Figure 5
Figure 5. Analysis of overlapped, differentially expressed genes in sequenced RNA from spironolactone- and prednisolone-treated mdx quadriceps myeloid cells.
(A) Heatmap of the 53 genes that are differentially expressed in both prednisolone treatment and spironolactone treatment conditions. Of the commonly differentially expressed genes, 45 genes were regulated in the same direction versus vehicle, while 8 were regulated in the opposite direction versus vehicle. Genes of interest are outlined in red boxes. (B) Transcripts per million of differentially expressed genes implicated in DMD pathology: Fn1, Hif1a, Thbs1, Fos, Dusp1, Ccl3, Vegfa, Per1, and Ccl4. Significant differences are from Benjamini-Hochberg adjusted P -values following DESeq2 analysis. *P ≤ 0.05.
Figure 6
Figure 6. Histology, myofiber degeneration, and fibrosis in spironolactone- and prednisolone-treated mdx diaphragms and quadriceps.
(A) Overall pathology (H&E) and staining for ongoing degenerating myofibers (IgG, green) and fibronectin (red) of diaphragm muscle sections from 4.5-week-old mdx mice treated with vehicle (VEH), spironolactone (SPR), or prednisolone (PRD) for 1 week shows active degeneration, inflammation, and fibrosis in dystrophic muscles. (B) Quantification of IgG+ fibers per 10 μm2. (C) Percent area of fibronectin staining in diaphragm shows improved dystrophic pathology with SPR but not PRD after 1 week of treatment (n = 7 SPR, n = 7 PRD, n = 8 VEH). (D) Staining for overall pathology (H&E), fibrosis (fibronectin, red) and colocalization of fibroblasts (vimentin, red), and myeloid immune cells (CD11b, green) at sites of injury in quadriceps muscle sections from 5.5-week-old mdx mice treated for 2 weeks with vehicle, SPR, or PRD. (E) Quantification of fibronectin staining in quadriceps muscle sections shows increased fibrosis with PRD treatment (n = 6 SPR, n = 6 PRD, n = 6 VEH). Scale bar: 100 μm. Statistics used were ANOVA with Dunnett’s test comparing each group with the vehicle (VEH). *P ≤ 0.05 and **P ≤ 0.01.
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