miR-29 family knock-down induces morphological and physiological changes in the heart of Zebrafish. (A) Representative images of Wild Type Zebrafish (left panel) and miR-29-sponge Zebrafish: view from outside (middle panel (+skin)) and with open chest (right panel (−skin)). Note the convexity in the cardiac region of miR-29 Sponge animal compared to Wild Type (black arrow) indicated by the white arrow. Calibration bar = 1 mm (B) Representative echocardiography of Wild Type (left panels) and miR-29-sponge (right panels) Zebrafish hearts showing end-diastolic area (EDA; first and third panel) and end-systolic area (ESA; second and fourth panel) Calibration bar = 1 mm. (C) Graph shows systolic area (black striped bars) and diastolic area (light gray bars) in Zebrafish hearts of Wild Type (n = 9) and miR-29-sponge Zebrafish (n = 13). (D) Graph shows Fractional Area Change (FAC) in Wild Type (black bar; n = 9) and miR-29-sponge (gray bar; n = 13) Zebrafish hearts. (E) Representative hematoxylin eosin staining in Wild Type (left panel) and miR-29-sponge (right panel) Zebrafish ventricles. Calibration bar = 100 µm. (D) *p < 0.05 Vs Wild Type.

miR-29 family knock-down associates with collagen deposition and DNA methylation increase in the Zebrafish heart. (A) Representative Fast Green Sirius Red staining of Wild Type (left panels) and miR-29-sponge (right panels) Zebrafish ventricles. Collagenous proteins are depicted in light purple and non-collagenous proteins in green. Magnification: 20x in first and third panel and 40x in second and fourth panel. Calibration bar = 25 µm (B) Collagen deposition quantification in sections derived from Wild Type (black circles; n = 8) and miR-29-sponge (gray squares; n = 8) Zebrafish hearts. (C) qRT-PCR analysis of collagen mRNAs in Wild Type (black circles; n = 4) and miR-29-sponge (gray squares; n = 4) Zebrafish hearts expressed as fold increase versus Wild Type samples. (D) Global DNA methylation quantification of 5mC in Wild Type (black circles; n = 3) and miR-29-sponge (gray squares; n = 3) Zebrafish hearts expressed as fold-change versus Wild Type samples. (E) qRT-PCR analysis of dnmt mRNAs in Wild Type (black circles; n = 4) and miR-29-sponge (gray squares; n = 4) Zebrafish hearts expressed as fold increase versus Wild Type samples. *p < 0.05 Vs Wild Type.