Therapeutic miR-21 Silencing Reduces Cardiac Fibrosis and Modulates Inflammatory Response in Chronic Chagas Disease

Figures

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  Figure 1. PCR array screening of selected miRNAs in Chagas disease. (A) Volcano plot of 88 miRNAs screened by PCR array in heart sample from chronic chagasic (n = 8) versus naïve (n = 4) mice (B) Volcano plot of 88 miRNAs screened by PCR array in serum samples obtained from chronic Chagas disease patients (n = 7) compared with healthy subjects (n = 3) (C) histogram of 45 deregulated miRNAs in heart sample from chronic chagasic (D) Venn diagram of PCR array showing five common miRNAs between human serum (pink) and mouse heart (blue) samples (E) histogram of 14 upregulated miRNAs in serum samples obtained from chronic Chagas disease patients. GenEX qPCR data analysis (Exiqon), p < 0.05; a cutoff value of 1.5-fold change-up and downregulated-was utilized in the analysis and miR-21 upregulation in both arrays is indicated by arrows.

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  Figure 2. In silico analysis of miRNA-mRNA interactions in Chagas disease. (A) miRNA expression in GEO datasets related to Chagas disease presented as -Log10 (p-value). Arrow indicates the miR-21 in all datasets. Datasets GSE75821 and GSE13791 referred to the results of in vitro studies that evaluated gene expression in host cells after T. cruzi infection. The dataset GSE41089 refers to a study that investigated cardiac mRNA expression during acute T. cruzi infection in C57BL/6. GSE17363 includes the results of mRNA expression in heart samples from C57BL/6 mice chronically infected with T. cruzi. GSE2596 shows mRNA expression profile in explanted hearts of patients with Chagas cardiomyopathy. The mRNA expression data and p-values were exported after being re-analyzed using the GEO2R. (B) Predicted consequential pairing of the target region (SPRY1 and STAT3) and conserved sites for miR-21-5p by TargetScanHuman.

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  Figure 3. MiR-21 and targets expression after T. cruzi infection in mouse cardiac fibroblasts. (A) MiR-21 expression in mouse cardiac fibroblasts 24 h after T. cruzi infection compared with non-infected control. (B) mRNA expression of miR-21 targets Spry1, Cadm1, and Stat3 24 h after T. cruzi infection. Tgfb1 mRNA expression was evaluated in vitro after T. cruzi infection in mouse fibroblasts. (C) Characterization of mouse and human cardiac fibroblasts by immunostaining for alpha-SMA and Vimentin markers (green), phalloidin (red), and nuclei stained with DAPI (blue). Scale bars = 50 µm. Expression analyses were performed by RT-qPCR (fold change to non-stimulated control), miRNA was normalized to miR-423-3p, and mRNA was normalized to HPRT/GAPDH. Data are expressed as mean ± SEM of three independent experiments performed. * p < 0.05, Student’s t-test.

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  Figure 4. Interplay of TGF-β signaling and miR-21 in mouse cardiac fibroblasts. (A) Scheme showing the TGFβ pathway in cardiac fibroblasts. (B) Increased expression of miR-21, Col1a1, Col1a2, Spry1, and Cadm1 in mouse cardiac fibroblasts, 24 h after stimulation with TGF-β1. (C) Expression of miR-21 and mRNA expression after stimulation with TGF-β1 and treatment with LNA-anti-miR-21, measured by RT-qPCR (fold change to non-stimulated control). miRNA expression levels were normalized to miR-423-3p, and mRNAs were normalized to HPRT/GAPDH. Results are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, Student’s t-test.

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  Figure 5. MiR-21 mediates fibrogenic activation in human cardiac fibroblasts. (A) Schematic representation of miR-21 overexpression by nucleofection of human cardiac fibroblasts with the expression vector and miR-21 inhibition by LNA anti-miR-21. (B) MiR-21, COL1A1, COL1A2, SPRY1, CADM1, and STAT3 expression levels 48h after nucleofection with miR-21 expression vector (pDNAmiR-21), measured by RT-qPCR (Fold Change to non-stimulated control). miRNA was normalized to miR-423-3p, and mRNAs were normalized to HPRT/GAPDH. (C) MiR-21, COL1A1, COL1A2, SPRY1, CADM1, and STAT3 expression levels after TGFβ1 stimulation or treated with LNA-anti-miR-21 for 24 h, measured by RT-qPCR (Fold Change to non-stimulated control). miRNA was normalized to miR-423-3p, and mRNAs were normalized to HPRT/GAPDH. Results are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.000.1 Student’s t-test.

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  Figure 6. Effects of the treatment with LNA-anti-miR-21 inhibitor in chronic chagasic mice. (A) Experimental design of the in vivo experiments. (B–E) Representative images of cardiac sections (ventricles) stained with Sirius Red. The following experimental groups were evaluated: naïve (B), saline (C), LNA scrambled (D), and LNA-anti-miR-21 (E). Scale bars = 100 μm. (F) Quantification of fibrosis area. (G) Heart samples from mice treated with LNA-miR-21 inhibitor were used to evaluate the miR-21 expression levels by RT-qPCR two weeks after treatment. (H) serum levels of cytokine TNF-α evaluated by ELISA. Data are represented as mean ± SEM. n = 8 per group. ANOVA with Bonferroni comparisons. * p < 0.05. ** p < 0.01. *** p < 0.001.

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  Figure 7. mRNA expression after treatment with LNA miR-21 inhibitor in chronic chagasic mice. Cardiac expression levels of Ptprc, Ifng, Tgfb1, Ctgf, Col1a1, Col1a2, Spry1, Cadm1, Mmp2, Mmp9 and Timp1 evaluated by RT-qPCR. Data are represented as mean ± SEM. n = 8 per group. ANOVA with Bonferroni comparisons. * p < 0.05. ** p < 0.01. *** p < 0.001.