MiR-145-5p modulates collagen production and fibroblast behaviour after cardiac injury partially via COL5A1

Figures

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  Fig. 1. Cryoinjury impairs cardiac function and alters transcriptome profiles in zebrafish hearts. a-b Cryoinjury impairs cardiac contractility at 7 dpi as manifested by the reduction in EF (a) and GLS (b) compared to baseline. The defect appears alleviated at 14 dpi. n = 11. c-d Principal component analyses of the mRNAome (c) and the miRNome (d). Data are presented as mean ± SD. One-way ANOVA with Tukey adjustment for multiple comparisons. * p < 0.05, ** p < 0.01

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  Fig. 2. Differentially expressed genes and significantly modulated molecular pathways in cryoinjured hearts at 14 dpi. a Heatmap shows the top 25 upregulated and downregulated genes ordered by adj. p-value in cryoinjured hearts at 14 dpi. Red colour represents upregulated genes while blue indicates downregulated genes. b-c Gene set enrichment analysis using GO categories indicates positive enrichment of GO-categories (b) and negative enrichment of GO-categories (c) in cryoinjured hearts. d Gene set enrichment analysis using the KEGG pathway browser. Red indicates positive enrichment; green indicates negative enrichment

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  Fig. 3. Differentially expressed miRNAs in cryoinjured hearts at 14 dpi and their predicted targets. a Heatmap shows the top 25 upregulated and 14 downregulated miRNAs ordered by adj. p-value (adj. p-value < 0.05) in cryoinjured hearts at 14 dpi. Red colour represents upregulated miRNAs while blue indicates downregulated miRNAs. b Predicted targets for the differentially expressed miRNAs (adj. p-value < 0.05) in cryoinjured hearts. The predicated targets are also differentially expressed in cryoinjured hearts (adj. p-value < 0.05). Orange colour represents the upregulated while light blue indicates downregulated miRNAs. Red colour shows positive correlation between miRNA and their target genes; dark blue indicates negative correlation. Arrowhead points the target genes of multiple miRNAs

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  Fig. 4. Validation of the most differentially expressed miRNAs and genes in cryoinjured hearts. a-c RT-qPCR analyses of the expression of miR-146a-5p (a), miR-181a-5p (b), and miR-145-5p (c) in zebrafish cryoinjured hearts compared to healthy controls. The results are presented as fold change. d-e RT-qPCR analyses of the expression of bmpr2a (d) and col5a1 (e) in zebrafish cryoinjured hearts compared to healthy controls. The results are presented as fold change. bmpr2a, bone morphogenetic protein receptor II a; col5a1, procollagen Vα1. RNA was extracted from 3 to 4 pooled hearts. The number of RNA samples used for RT-qPCR analyses: control n = 3; infarct n = 3. f-g RT-qPCR analysis of miRNA-145-5p expression in the LV of ICM patients (f) and human primary cardiac fibroblast (HCF) treated with TGF-β1 or TGF-β2 for 24 hours (g) compared to respective controls. Controls n = 6; ICM n = 7. h Representative immunoblot and quantification of COL5A1 in the LV of ICM patients compared to controls. Controls n = 6; ICM n = 6. Data are presented as mean ± SD. Two-sample t-test. One-way ANOVA with Tukey adjustment for multiple comparisons. * p < 0.05, ** p < 0.01

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  Fig. 5. miR-145-5p regulates collagen expression and cardiac fibroblasts differentiation, proliferation, and migration. a Representative immunoblot and quantification of αSMA in HCF either treated with miR-145-5p inhibitor or inhibitor control. b RT-qPCR analysis of ACTA2 transcript in HCF either treated with miR-145-5p inhibitor or inhibitor control. c Representative immunoblot and quantification of COL1A1 in HCF either treated with miR-145-5p inhibitor or inhibitor control. d Representative immunoblot and quantification of αSMA in HCF either treated with miR-145-5p mimic or mimic control. e Representative immunoblot and quantification of COL1A1 in HCF either treated with miR-145-5p mimic or mimic control. f-g Representative immunocytochemical staining of αSMA (in green) and vimentin (in red) in HCF treated with miR-145-5p inhibitor (f) or miR-145-5p mimic (g) and their respective controls. Graph indicates the quantification of αSMA+ cells relative to DAPI counts. Arrow indicates αSMA+ fibroblast. Scale bar: 50 µm. h-i Real-time proliferation profiles of HCF transfected with miR-145-5p inhibitor (h), miR-145-5p mimic (i), or corresponding controls. Prior to the xCELLigence assays, cells…

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  Fig. 6. Identification of COL5A1 as a target gene of miR-145-5p. a-b RT-qPCR analyses of COL5A1 expression in HCF either treated with miR-145-5p mimic (a) or inhibitor (b) compared with relevant controls. c-d Representative immunoblot and quantification of COL5A1 in HCF either treated with miR-145-5p mimic (c) or inhibitor (d) compared with relevant controls. e, Schematic presentations of the COL5A1 3′UTR with predicted miR-145-5p binding sites. f-g The relative firefly luciferase activity was normalized to the Renilla luciferase signal for each sample. HEK cells were transfected with pEZX-MT06-COL5A1-3UTR carrying targeting region 1 (f) or region 2 (g) either alone or with mimic control, or miR-145-5p mimic, for 24 hours. Data are presented as mean ± SD. Two-sample t-test. One-way ANOVA with Tukey adjustment for multiple comparisons. * p < 0.05, ** p < 0.01, *** p < 0.001

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  Fig. 7. COL5A1 regulates collagen composition, fibroblast dynamics, and cardiac fibrotic scar resolution. a Cytoscape analysis of collagen network and their distribution in cardiac cells. Red colour shows higher relative expression level; blue indicates lower level; grey indicates undetected gene in the source data. b RT-qPCR analysis of COL5A1 expression in HCF either treated with COL5A1 GapmeR DR2 (COL5A1 G2) alone or together with miR-145-5p inhibitor (miR-145 inh.), compared to negative control (NC). c Representative immunoblot and quantification of COL5A1 in HCF either treated with COL5A1 G2 alone or together with miR-145-5p inhibitor, compared to negative control. d RT-qPCR analysis of COL1A1 expression in HCF either treated with COL5A1 G2 alone or together with miR-145-5p inhibitor, compared to negative control. e Representative immunoblot and quantification of COL1A1 in HCF either treated with COL5A1 G2 alone or together with miR-145-5p inhibitor, compared to negative control. f Real-time proliferation profiles of HCF transfected with COL5A1 G2 alone or together with miR-145-5p inhibitor, compared to negative control. g Real-Time migration profiles of HCF transfected with COL5A1…

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  Fig. 8. miR-145-5p potentially regulates integrin-mediated pathways independent of COL5A1 in human cardiac fibroblast. a-b RT-qPCR analyses of ITGβ3 (a) and ITGβ5 (b) expression in HCF treated with COL5A1 G2, compared to NC. c Representative immunoblot and quantification of ITGβ3 in HCF treated with COL5A1 G2, compared to NC. d Representative immunoblot and quantification of ITGβ5 in HCF treated with COL5A1 G2, compared to NC. e-f RT-qPCR analyses of ITGβ3 (e) and ITGβ5 (f) expressions in HCF treated with miR-145-5p inhibitor compared with inhibitor control. g-h Representative immunoblot and quantification of ITGβ3 (g) and ITGβ5 (h) expressions in HCF either treated with cilengitide alone or in combination with miR-145-5p inhibitor, compared to NC. i Representative immunoblot and quantification of pSMAD2/3 relative to SMAD2/3 in HCF either treated with cilengitide alone or in combination with miR-145-5p inhibitor, compared to NC. The expressions of pSMAD2/3 and SMAD2/3 were normalized to tubulin in the respective gels, then the phosphorylation of SMAD2/3 was quantified by normalizing it to SMAD2/3. j Real-time proliferation profiles of HCF treated with cilengitide alone or together with…