A miR-200b–Filamin A axis drives epicardial contribution to cardiogenesis

Figures

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  Fig. 1. miR-200b is expressed in a subset of epicardial cells during EPDCs emergence in the developing heart. (A) Transcript levels of miR-200b, miR-1 and miR-133a in the developing mouse heart from E10.5 to E16.5. Error bars indicate SD. n = 3; each point represents averaging of three technical replicates. Statistical analysis was performed using a one-way ANOVA followed by Dunnett’s multiple comparisons test. (B) Representative FISH images showing miR-200b expression across different epicardial regions at high magnification (63 × objective) in embryonic heart sections from WT1Cre-YFP mouse embryos at E12.5, E14.5, and E15.5. To provide anatomical context for the high-magnification images, a representative reconstruction of a whole E12.5 heart, generated from low-magnification images (4 × objective), is included. In the low-magnification image, three boxed regions represent the areas from which high-magnification images were acquired at E12.5, E14.5, and E15.5, corresponding to panels B′, B″, and B. The basement membrane is delineated by a dotted line marking the boundary between the epicardial layer (YFP⁺) and the underlying subepicardium and outer myocardium containing EPDCs (YFP⁺).…

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  Fig. 2. miR-200b gain- and loss-of-function modifies EPDCs generation from the embryonic epicardium. (A) Scheme of miR200b gain- and loss-of-function experiments, by transfection of premiR-200b and antimiR-200b molecules, in embryonic heart explants isolated from WT1Cre-YFP mice. (B) Transcript levels of miR-200b in the embryonic heart explants after premiR-200b and antimiR-200b transfections. Error bars indicate SD. n = 3. Each point represents the mean of three technical replicates. Statistical analysis was performed using a one-way ANOVA followed by Dunnett´s multiple comparisons test. (C) Representative images of epicardial and epicardial-derived cells (YFP+ cells) in the embryonic heart explants transfected with premiR-200b, antimiR-200b and scramble-control molecules. The boundary between the epicardium and the myocardium has been delineated with a dotted line. White arrows indicate EPDCs. Scale bars: 50 μm. (D) Quantifications of the percentage of epicardial-derived cells (YFP+ cells) internalized into the myocardium in the embryonic heart explants transfected with premiR-200b, antimiR-200b and scramble control molecules. Error bars indicate SD. n = 4–14. Each point displayed in…

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  Fig. 3. miR-200b alters mitotic spindle orientation in the embryonic epicardium. (A) Scheme for symmetric and asymmetric cell division analysis criteria in the epicardial cells. The polar chart illustrates how the orientations of epicardial cell spindles were quantified. The basement membrane was considered 0° and is represented by the black dotted line. Each line corresponds to the spindle orientation of a dividing epicardial cell. Divisions with spindle orientations between 30° and 330° were classified as symmetric, whereas those between 60° and 120° were classified as asymmetric. (B) Representative image of symmetrical (dashed arrow) and asymmetrical cell division (arrow) in the epicardial layer. Scale bars, 25 μm. The basement membrane is indicated by a dotted line. (C) Ratio of epicardial cell spindle orientation in the embryonic epicardium, from E12.5 to E15.5. Error bars indicate SD. n = 3. (D) Scheme of gain- and loss-of-function experiments, by premiR-200b and antimiR-200b molecules transfection, in embryonic heart explants. (E) Transcript levels of miR-200b in embryonic transfected-heart explants. Error bars indicate SD. n = 3. Each point represents the mean of three technical…

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  Fig. 4. miR-200b targets FLNA in MEC1 epicardial cells. (A) Schematic representation of miR-200b target identification in MEC1 cell line by RNA pull-down. (B) miR-200b-binding mRNAs in MEC1 epicardial cells obtained by RNA-seq analyses after RNA pulldown. (C) Luciferase reporter assay assessing post-transcriptional regulation of the FLNA 3′UTR by miR-200b. Relative luciferase activity (arbitrary units, a.u.) was measured in 3T3 cells transfected with the FLNA 3′UTR construct alone (FLNA 3′UTR), co-transfected with miR-200b (FLNA 3′UTR + miR-200b), or co-transfected with a mutated 3′UTR construct and miR-200b (FLNA 3′UTR mut + miR-200b). Error bars indicate SD.n = 3. Statistical analysis was performed using the unpaired Student’s T-test. (D) Scheme of premiR-200b and antimir-200b transfection in MEC1 cells. (E) Transcript levels of miR-200b in MEC1 cells after premiR, antimiR and scramble-control transfections. Error bars indicate SD. n = 3. Each point represents the mean of three technical replicates. Statistical significance was determined using a one-way ANOVA followed by Dunnett’s multiple comparisons test. (F) Transcript levels of Flna in premiR-200b, antimiR-200b and scramble…

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  Fig. 5. miR-200b regulates FLNA expression in the epicardial layer. (A) Scheme of gain-and loss-of-function experiments, by premiR-200b, antimiR-200b and scramble-control molecules, in embryonic heart explants. Co-electroporation with a plasmid expressing GFP allowed us to trace which cells were successfully transfected. (B) Transcript levels of miR-200b in embryonic heart explants from WT1Cre-YFP mice after premiR, antimiR and scramble-control transfections. Error bars indicate SD. n = 3. Each point represents the mean of three technical replicates. Statistical analysis was performed using a one-way ANOVA followed by Dunnett´s multiple comparisons test. (C) Immunostaining for FLNA in electroporated heart explants. The boundary between the epicardium and the myocardium has been delineated with a dotted line. For a clearer view, FLNA-staining is shown in both magenta and gray scale. Scale bars, 50 μm. (D) Quantification of corrected FLNA fluorescence intensity. Error bars indicate SD. n = 3–5. Each point displayed in the graph represents a single quantified image obtained from independent biological samples. Each colour represents an independent biological replicate. Statistical analysis…

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  Fig. 6. FLNA loss-of-function compromises the number of asymmetric cell divisions in the epicardium. (A) Schematic overview of the experimental workflow. Hearts from mouse embryos at E12.5 were isolated and transfected with scramble or FLNA siRNA after 24 h of explant cultures. Cy5 co-transfection allowed us to trace which cells were successfully transfected. After 24 h of co-transfection, cultured embryonic heart explants were collected and quantitative real-time PCR (RT-qPCR) as well as Aurora B immunostaining were performed. (B) Quantification of FLNA mRNA expression levels in Scramble and FLNA siRNA transfected embryonic heart explants. Error bars indicate SD. n = 3. Statistical analysis was performed using the unpaired Student’s T-test. (C) Representative images of symmetric (dashed arrow) or asymmetric (arrows) cell division, Aurora B staining, in epicardial cells of heart sections from siFLNA transfected explants. Aurora B staining was used to define spindle orientation and GFP-expression to identify siFLNA transfected cells. The basement membrane is delineated by a dotted line marking the boundary between the epicardial layer and the subepicardium and outer myocardium. The…

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  Fig. 7. miR-200b expression is re-activated after MI in the adult heart. (A) Schematic overview of the experimental workflow. Hearts from infarcted and sham-operated mice were collected at different time points (days 1, 3, 7, and 14 post-infarction) for RNA isolation and quantitative real-time PCR (RT- qPCR) assays. (B) Quantification of miR-200b expression in bulk cardiac tissues from sham-operated and infarcted hearts at multiple time points after MI. A significant increase in miR-200b expression was observed in the infarcted region at day 7 post-infarction. Statistical analysis was performed using Fisher’s Least Significant Difference (LSD) test. (C) Schematic illustration of the experimental workflow. Serum samples were collected from infarcted and non-infarcted (control) human patients, and RNA isolation and quantitative real-time PCR (RT-qPCR) were performed to evaluate the expression of hmiR-200b in peripheral blood. (D) Box plot showing hmiR-200b expression levels in control and infarcted human serum samples. A significant reduction in miR-200b expression was observed in infarcted patients compared to controls. Statistical analysis was performed using the unpaired Student’s…