Figure 1.
Low power images of excised cultured hearts (days 0–5).
Upper panel (A) showing examples of excised hearts maintained in culture for 0, 3 and 5 days. Lower panel (B) shows example traces from Video-edge detection method displaying movement of the ventricle wall which was used to assess heart rate and ventricle function at days 0,3 and 5 in culture.
Figure 2.
Functional assessment of isolated hearts in culture (days 0–5).
Heart rate and ventricle function assessed using video edge detection of wall motion during spontaneous beating (n = 6 hearts over 5 days, mean±sem). Data analysed by 2 way ANOVA and Bonferroni correction for multiple comparison show a clear interaction of all parameters with time over the 5 days in culture and a clear interaction with response to isoproterenol (*p<0.05).
Figure 3.
Heart mould for high throughput histology.
(a) 3D render of cutaway top (white) and bottom part (red) of the designed heart mould. (b) Render of single well from above with superimposed dimensions. (c) 2D cutaway of a single well of the heart mould showing dimensions. (d) Top and bottom sections of heart mould machined from acrylic. (e) Top down view of single agar well with zebrafish heart. (f) Cut-away side view of single well showing heart and sectioning plane. (g) Example slide with grid of hearts with histology-dye borders showing arrow row and column markers. (h) High-magnification of single H&E stained heart. V, ventricle. A, atrium. B.a., bulbus arteriosus.
Figure 6.
Apoptosis in isolated cultured zebrafish hearts (TUNEL staining).
Apoptotic nuclei (green) and nuclear DAPI staining (blue) in zebrafish hearts (assessed by TUNEL staining) maintained in culture for 0, 1, 3 and 5 days showing a marked increase in the number of apoptotic bodies at day 3 which is maintained at day 5.
Figure 4.
Histological analysis of isolated hearts.
Haematoxylin and Eosin (H&E; a–i) and Masson’s trichrome staining (j–r) of hearts at 0, 3 and 5 days in culture. Higher magnification images (g, h, i) show normal cellular architecture at day 0 and day 3 with significant changes at day 5.
Figure 5.
Immunohistology of isolated cultured hearts.
Alpha-actinin immunostaining showing well preserved sarcomere patterns in cardiomyocytes of cultured hearts at day 0 and day 3 with loss of this typical pattern by day 5 in culture. (red is sarcomeric alpha-actinin (mouse, clone EA-53, Abcam), blue is DAPI (Serva Heidelberg)).