Failure to resolve inflammation contributes to juvenile onset cardiac damage in a mouse model of Duchenne muscular dystrophy

Figures

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  Fig. 1. Histopathology associated with disease onset in juvenile D2-mdx hearts. Images show hearts harvested from juvenile (6 ± 0.5 wk) D2-mdx and B10-mdx mice at disease onset. A, B Whole tissue images with matched orientation of D2-mdx (A) and B10-mdx (B) hearts showing ventricular and atrial fibro-calcified damage. C, D Cross-sectional images of juvenile D2-mdx and B10-mdx hearts through the ventricular lumen, stained for histological features by H&E (C), and for fibrosis by Sirius Red (D). Arrowheads mark areas of fibrosis. E, F Image (E) and quantification (F) showing a portion of heart cross-section from juvenile D2-mdx and B10-mdx hearts, showing damaged tissue areas characterized by the presence of interstitial fibrosis, inflammatory cells and damaged cardiomyocytes. G, H Image (G) and quantification (H) showing a zoomed-in portion of heart cross-section from juvenile D2-mdx and B10-mdx hearts labeled with Sirius Red to mark fibrotic tissue area in hearts from juvenile D2-mdx and B10-mdx mice. Data represent median ± IQR from n = 10-12 hearts per cohort, with statistical analyses performed using non-parametric Mann–Whitney test; **p < 0.01, ***p < 0.001. Refer to Supplementary…

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  Fig. 2. RNAseq analysis at disease onset in D2-mdx and age-matched B10-mdx hearts. Juvenile D2-mdx and B10-mdx hearts at disease onset (6 wk ± 0.5 wk) were analyzed by bulk tissue RNAseq. A Dimensionality reduction of whole transcriptomic data via PCA (n = 3−4 hearts/genotype) to assess sample clustering and inter-/intra-sample variance. B Differential gene expression analysis depicted via heatmap plot of 2719 DEGs observed between D2-mdx (blue) and B10-mdx (red), with 1586 genes upregulated for D2-mdx and 1133 genes upregulated for B10-mdx. Expression is z-score values of variance-stabilizing transformation (VST) normalized data. C Gene Ontology (GO) analysis performed using Cytoscape and EnrichmentMap plugins to identify networks of related GO terms groups found upregulated (red dots) in juvenile D2-mdx hearts relative to B10-mdx. Pink clusters refer to inflammatory-related GO terms, while blue clusters refer to extracellular matrix-related GO terms. D, E Boxplots showing VST normalized gene expression levels for top 20 differentially expressed inflammation-related (D; GOBP:Inflammatory Response) and extracellular matrix-related (E; GOBP:External Encapsulating Structure) transcripts…

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  Fig. 3. Targeted analysis of inflammatory response at disease onset in D2-mdx and B10-mdx hearts. A qRT-PCR analysis of a distinct cohort of D2-mdx and B10-mdx hearts to assess the expression of inflammatory genes identified by RNAseq analysis cohort to be differentially expressed. Transcripts include top dysregulated genes involved in leukocyte activation, migration and chemotaxis and regulation of inflammatory response and leukocyte-mediated immunity (Ccl3, Ccl8, Stab2, Adam8, Trem2, Il7r). Relative gene expression values normalized to internal Hprt transcript levels. B qRT-PCR analysis of a distinct cohort of D2-mdx and B10-mdx hearts to assess inflammatory genes that show broad dysregulation of neutrophil and macrophage response in juvenile D2-mdx hearts (Il1b, Lgals3, Arg1, Fpr1, Fpr2, Anxa1). Relative gene expression values normalized to internal Hprt transcript levels. C, D Images showing immunostaining for pan-macrophage marker, F4/80 (green), and pro-inflammatory, pathogenic macrophage marker, GAL-3 (red), in juvenile D2-mdx (C) and B10-mdx (D). Data represent median ± IQR from n = 7−9 hearts per cohort. Statistical analyses performed using non-parametric Mann–Whitney test; **p…

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  Fig. 4. Targeted analysis of extracellular matrix remodeling response at disease onset in D2-mdx and B10-mdx hearts. A qRT-PCR analysis of a distinct cohort of D2-mdx and B10-mdx hearts to assess the expression of extracellular matrix-associated genes involved in matrix organization/re-organization (Fn1, Col1a1, Itgax, Spp1, Mmp12, Timp1) that are identified to be dysregulated by the RNAseq cohort. Relative gene expression values normalized to internal Hprt transcript levels. B-C. Images showing extracellular matrix distribution visualized using wheat germ agglutinin (WGA, pink) within, and surrounding, areas of cardiac damage in juvenile D2-mdx (B), and B10-mdx (C). B’, C’ Zoom of the dotted area from whole cross-sectional images showing immunostaining for COL1A1 within the extracellular matrix shows increased COL1A1 expression in damaged D2-mdx hearts (B’), relative to B10-mdx (C’), indicative of early-onset endomysial fibrosis. Data represent median ± IQR from n = 7−9 hearts per cohort. Statistical analyses performed using non-parametric Mann–Whitney test; **p < 0.01, ***p < 0.001. For age-matched WT controls, refer to Supplementary Fig. 4.

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  Fig. 5. Pro-resolving therapy to mitigate cardiac disease onset in juvenile D2-mdx. A Schematic describing the inflammatory response following cardiac injury in health (black trace) or dystrophic (red trace), showing acute versus chronic inflammation respectively. Use of anti-inflammatory drug (purple trace) lowers inflammatory response blunting inflammation, instead use of pro-resolving therapy (green trace) does not impact the onset of inflammation but helps clear inflammation preventing the inflammation to become chronic. B Schematic detailing the pre-clinical testing of pro-resolving drug, BMS-986235 (6.0 mg/kg, 3 wk daily administration) in D2-mdx mice (n ≧6) just prior to disease onset (18-19 days old). C Whole tissue images of the matched orientation of hearts showing ventricular and atrial fibro-calcified damage in saline or BMS-986235-treated D2-mdx mice. D Image showing cross-section of D2-mdx heart stained for histological features by H&E from mice treated with saline or BMS-986235. E Images showing cross-section of D2-mdx heart immunostained for pan-macrophage marker, F4/80 (red) and counterstained with WGA (green) and DAPI (blue) to mark the ECM and nuclei, respectively. F…