Caveolin-1 is expressed in the endothelium, endocardium and epicardium of the intact and injured adult zebrafish heart. (a) Immunofluorescence staining of Cav1 and Tropomyosin (cardiomyocytes) in an intact Tg(fli1a:GFP) heart. ba, bulbus arteriosus; v, valves. (b–c′′) Cav1 immunoreactivity in the epicardium (arrows) overlaps with GFP in the endothelium (asterisks) and endocardium (arrowheads). Cav1 is also expressed in the zone between the cortical and trabecular cardiomyocytes (insert in b′). (d–f′) Cav1 immunostaining in 7 dpci Tg(wt1b:GFP) heart. The dashed area in (d) marks the injured area; Cav1 is expressed in the activated epicardium (e–f′ brackets) and endocardium (f, arrows) upon injury. (g–h′) Immunolabelling of Cav1 in a 7 dpci Tg(fli1a:GFP) heart. (h–h′) Arrows indicate Cav1⁺ endocardial cells. Scale bars: 100 μm in (a), (d), (e) and 50 μm in other panels. (i) qPCR analysis of caveolae-related genes during regeneration. Mean ± s.d., Brown-Forsythe and Welch ANOVA tests, *P < 0.05, **P < 0.01.

Generation of cav1-KO by CRISPR/Cas9 editing and transcriptional analysis. (a) Schematic representations of the two cav1 transcripts—cav1a and cav1b. Target site of the guide RNA is indicated by a red arrowhead. ex, exon. (b) Identified genetic mutations. Red dashed lines indicate deletions, red uppercase letters insertions, and black lowercase letters silent mutations. bp, base pairs. (c) Cav1 domain organisation and the predicted effect of the mutations on the protein. The novel amino acids are in red, x indicates a stop codon. (d) qPCR analysis of caveolae-related genes in two-day post fertilisation embryos. Mean ± s.d., t-test, **P < 0.01. (e–j) Immunostaining of Cav1a in 7 dpci hearts. (f, g, i, j) Higher magnification of the dashed boxes in (e) and (h), respectively. (k–p) Cav1 (Cav1 and Cav1b) immunolabelling of 7 dpci hearts. (l, m, o, p) Higher magnification of the dashed boxes in (k) and (n), respectively. Dotted areas in (h) and (n) mark the valves in the mutants. Scale bars: 100 μm in (e), (h), (k), (n); 50 μm in other panels.

Loss of caveolae in cav1^cn100 hearts. TEM images of cav1^+/+ (a, a′) and cav1^cn100 (b, b′) endothelial cells from the coronary vasculature. (a′, b′) Higher magnification of the dashed areas in (a) and (b). Arrowheads indicate membrane-bound caveolae. Scale bars: 1 μm in (a), (b); 0.5 μm in (a′), (b′). (c) Caveolae number per μm² of endothelial cell. n_WT = n_cn100 = 4, t-test, **P < 0.01.

Heart regeneration is unaffected in cav1^cn100 mutants. (a–j) cav1^+/+ and cav1^cn100 hearts were cryoinjured and harvested 30 (a, b), 60 (d, e) or 90 dpci (g, h), and processed for AFOG staining, which labels collagen in blue, fibrin in red and healthy myocardium in brown. (i) heterozygous cav1^+/cn100 heart, 90 dpci. (c, f, j) Quantification of the injury site. Injuries were quantified as a percentage of the damaged tissue (collagen and fibrin) to the total area of the ventricle. 30 dpci n_WT = n_cn100 = 10; 60 dpci n_WT = n_cn100 = 10; 90 dpci n_WT = 9, n_cn100 = 12, t-test. Scale bars 250 μm.

Cardiomyocyte proliferation is transiently reduced upon cryoinjury in cav1^cn100 hearts. (a, b) Immunolabelling of 7 dpci cav1^+/+ and cav1^cn100 hearts for BrdU, MEF2 and MF20. (a′, b′) Magnifications of the dashed areas in (a) and (b). (c) Cardiomyocyte proliferation rate was measured by quantifying the BrdU⁺/MEF2⁺ nuclei to the total cardiomyocyte number in a 100 μm radius around the injured area. CM, cardiomyocytes. n_WT = 5, n_cn100 = 6, t-test, **P < 0.01. (d, e) Immunostaining of 14 dpci cav1^+/+ and cav1^cn100 hearts for BrdU, MEF2 and MF20. (d′, e′) Magnifications of the dashed areas in (d) and (e). (f) Quantification of cardiomyocyte proliferation at 14 dpci. n_WT = 7, n_cn100 = 4, t-test. Scale bars: 100 μm in (a), (b), (d), (e); 10 μm in (a′), (b′), (d′), (e′).

Impaired cardiac function and stiffer heart tissue in caveolae-deprived cav1^cn100 mutants. (a) Quantification of ejection fraction in cav1^+/+ and cav1^cn100 hearts. n_WT = 22, n_cn100 = 20. (b) Heart rate measurements in cav1^+/+ and cav1^cn100 animals. n_WT = 23, n_cn100 = 20. t-test, *P < 0.05. (c) AFM set-up. Cartoon made with Adobe Illustrator CC2018 (www.adobe.com). (d, e) Images of the cantilever (arrowhead), the laser beam on top of the cantilever (dashed circle, also in c) and the ventricle (asterisk). Images were taken with an inverted optical microscope (AXIO Observer D1; Carl Zeiss, Germany, see Methods). (f) Biomechanical characterization of cav1^+/+ and cav1^cn100 ventricles as measured by AFM-force spectroscopy and expressed in Young’s moduli. n_WT = 9, n_cn100 = 8, t-test, ***P < 0.001. (g) Force-distance graph for indentation and retraction of the cantilever over the ventricular surface.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.

ZFIN is incorporating published figure images and captions as part of an ongoing project. Figures from some publications have not yet been curated, or are not available for display because of copyright restrictions.