Circulating miRNAs as Potential Biomarkers Associated with Cardiac Remodeling and Fibrosis in Chagas Disease Cardiomyopathy

Figures

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  Figure 1. MicroRNA validation in serum of chronic Chagas disease subjects and healthy controls. Healthy controls (CTL), n = 10, Indeterminate form (IF), n = 10, and chronic Chagas cardiomyopathy (CCC), n = 28, were evaluated by RT-qPCR, (A) miR-19a-3p, (B) miR-21-5p and (C) miR-29b-3p, (D) miR-30a-5p and (E) miR-199b-5p, normalized by miR-93 and miR-16. One-way ANOVA followed by Newman-Keuls post-test. * p < 0.05.

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  Figure 2. Receiver operating characteristic (ROC) curve for the prognosticating Chagas disease progression. ROC analysis of miRNA expression in serum from chronic Chagas disease subjects, indeterminate form (IF) vs chronic Chagas cardiomyopathy (CCC): (A) miR-19a-3p, (B) miR-21-5p, (C) miR-29, and (D) miR-199b-5p expression, measured by RT-qPCR. The miRNA values were standardized for statistical analysis. The area under the curve (AUC) with 95% CI and P value were represented in the figure. p < 0.05 indicate statistical significance.

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  Figure 3. Histological and miRNA analysis in explanted hearts from chronic Chagas disease subjects. Left ventricle sections stained with H&E showing (A) a normal area of myocardium with minimal myocyte hypertrophy; (B) interstitial fibrosis and moderate myocyte hypertrophy; (C) severe myocyte hypertrophy, with the presence of enlarged and pleomorphic nuclei, varying in shape and size. The fibers are random and there is focal interstitial inflammation (top left corner); (D) multifocal inflammation characterized by accumulation of plasma cells and mature lymphocytes. The nuclei of myocytes are mildly enlarged and pleomorphic (moderate myocyte hypertrophy), and there is mild interstitial fibrosis. Bars = 500 μm; (E) miR-19a-3p, miR-21-5p, miR-29b-3p, miR-30a-5p, miR-199b-5p and miR-208a-3p evaluation by RT-qPCR, using 2−∆∆Ct method and normalized with miR-423-3p. Data represent the mean ± SEM. Controls (CTL), n = 8, and chronic Chagas cardiomyopathy (CCC), n = 9, analyzed using Student’s t-test, *** p < 0.001, ** p < 0.01.

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  Figure 4. Assessment of miRNA expression in human cardiac fibroblasts after stimulation with TGFβ. (A) Experimental design. (B) Validation of human cardiac fibroblasts markers by immunostaining for DDR2 (A, green), α-SMA (B, green) and vimentin (C, green). In all samples, the cytoskeleton was stained with phalloidin (red) and nuclei were stained with DAPI (blue). Scale bars = 50 μm; (E) Staining for collagen type I (red) and nuclei were stained with DAPI (blue) after 24 h of incubation with TGF-β. Scale bars = 20 μm; (F) Quantification of COL1A1 expression by measurements of median fluorescence intensity and (G) gene expression by RT-qPCR. (H) Expression levels of miR-21-5p in human cardiac fibroblasts stimulated or not with TGF-β1, evaluated by RT-qPCR, normalized with miR-423-3p and represented by fold change expression. CTL = unstimulated cardiac fibroblasts. TGF-β = cardiac fibroblasts stimulated with TGF-β. Values represent the mean ± SEM of n=3 replicates group and were evaluated using Student’s t-test. * p < 0.05.

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  Figure 5. Validation of an in vitro hypertrophy model using human induced pluripotent stem cell-derived cardiomyocytes stimulated with endothelin-1. (A) Experimental design. (B–G) Characterization of hiPSC-CM. Morphology of hiPSCs visualized by phase-contrast microscopy at differentiation D0 (B) and D14 (C). (D) Differentiation efficiency measured by flow cytometry analysis of cTNT+ cells. Histograms show differentiated cells on the left (red histogram) and undifferentiated hiPSCs on the right (grey histogram). Scale bars = 100 μm. (E–G) Immunostaining for cardiomyocyte markers sarcomeric alpha-actinin (E), cardiac troponin T and GATA-4 (F), and MF-20 (G). Scale bars = 50 μm. (H) Increased BNP expression at the mRNA level, evaluated by RT-qPCR and at the protein level in the (I) culture supernatant 24 h after ET-1 incubation. (J) Cardiomyocyte area measured by quantification of cTNT staining. CTL = unstimulated hiPSC-CMs, ET1 = hiPSC-CMs stimulated with ET-1. Values represent the mean ± SEM of n = 3 replicates group and were evaluated using Student’s t-test. * p < 0.05 and *** p < 0.001.

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  Figure 6. Increased miRNA expression after hypertrophic stimulus with endothelin-1 in hiPSC-CM. Expression levels of miR-19a-3p (A), miR-21-5p (B), miR-29b-3p (C) and miR-199b-5p (D) in hiPSC-CM stimulated with ET-1, evaluated by RT-qPCR. CTL = unstimulated hiPSC-CMs, ET1 = hiPSC-CMs stimulated with ET-1. Values represent the mean ± SEM of n = 3 per group. Data were analyzed using Student’s t-test. * p < 0.05; ** p < 0.01.