Fibulin-3 Deficiency Protects Against Myocardial Injury Following Ischaemia/ Reperfusion in in vitro Cardiac Spheroids

Figure 1. Fib-3 KO CSs form faster than WT CSs. (A) Representative images of WT and Fib-3 KO CSs at day 4. (B) Statistical analyses of CS formation in WT vs. Fib-3 KO CSs. Unpaired t-test, p < 0.0001 = ****. Error bars represent the mean ± SD (n = 384).

Figure 2. Fib-3 KO CS demonstrate reduced CD31-positive endothelial cells compared to WT CSs. (A, B) Collapsed Z-stacks of confocal images of WT and Fib-3 KO CS stained with antibodies against CD31 for endothelial cells (green), cTNT for cardiomyocytes (red) and Hoechst for nuclei (blue). (C, D) 3D rendering analyses of confocal images of WT (A) and Fib-3 KO CS (B), respectively. (E) 3D representation of endothelial cell network formation in WT and Fib-3 KO CS (blue dots represent foci of the endothelial cell network formed within each CS). (F) Statistical analyses of expression of cTNT normalized against Hoechst stain in WT and Fib-3 KO CSs, (n = 5). (G–I) Statistical analyses of endothelial cell network length, area and volume in WT and Fib-3 KO CSs. Unpaired t-test, p>0.05= ns, p < 0.01 = **. Error bars represent the mean ± SD (n>32).

Figure 3. Establishment of hypoxic/normoxic damage in Fib-3 KO and WT CSs. (A) Epifluorescent images of a representative CS treated with Image-iT (become fluorescent red in lower oxygen conditions) that was pre-incubated at 5% O2 for 24 h (T = 0), and then exposed to 0% O2 for 20 h (T = 20) and 5% O2 for additional 17 h (T = 37). (B) Statistical analysis of changes i n Image-iT fluorescence between 5% (T = 0), 0% (T = 20) and 5% (T = 37) between WT and Fib-3 KO CSs. One-way ANOVA with Tukey post-hoc test was used f or comparison of all groups, p < 0.05 = *, p < 0.01 = **, p < 0.001 = ***, and p>0.0001 = ****. Error bars represent the mean ± SD (n = 3).

Figure 4. Fib-3 KO CSs are less sensitive to changes in oxygen concentrations compared with WT CSs. (A–L) Representative images of control and I/R WT and Fib-3 KO CSs stained with calcein-AM (green), ethidium homodimer (red) and Hoechst stain (blue), staining live, dead, and total cells, respectively. (A–D) stained with Hoechst, (E–H) with calcein-AM, and (I–L) and ethidium homodimer. (M) Statistical analyses of toxicity ratios for control and I/R CSs. Two-way ANOVA with Sidak's multiple comparison test, p>0.05= ns and p < 0.01 = **. Error bars represent the mean ± SD (n>6).

Figure 5. I/R conditions decrease contraction frequency and fractional shortening in CSs. Statistical analysis of contractile frequency (A) and fractional shortening (B) of control and I/R CSs from both WT and Fib-3 KO mouse optimized under Olympus microscope of Ionoptix system. One-way ANOVA with Tukey post-hoc test was used for comparison of all groups, p > 0.05 = ns, p < 0.05 = *, p < 0.001 = *** and p < 0.0001 = ****. Error bars represent the mean ± SD (n > 5).

Figure 6. Fib-3 KO and WT CSs differentially respond to changes in oxygen concentrations. Heat map of the differential expression of cluster of cardiovascular genes of WT and Fib-3 KO I/R CSs moving from right to left. The color bar on the right side indicates the intensity of expression.

Figure 7. Changes in cardiac remodeling genes in WT and Fib-3 KO CSs following I/R injury. Relative expression of Ace, Mmp13 and Ren1 in WT and Fib-3 KO CSs. Two-way ANOVA with Sidak's multiple comparison test, p > 0.05 = ns and p < 0.0001= ****. Error bars represent the mean ± SD (n = 4).

Figure 8. Changes in apoptosis regulating genes in WT and Fib-3 KO CSs following I/R injury. Relative expression of Anxa4, Ccl11, Ccl2, Maoa, Nppa, Nppb and Npr1 in WT and Fib-3 KO CSs. Two-way ANOVA with Sidak's multiple comparison test, p > 0.05 = ns and p < 0.0001= ****. Error bars represent the mean ± SD (n = 4).

Figure 9. Changes in fibrotic genes in non-infarcted WT and Fib-3 KO CSs. Relative expression of Col11a1, Col1a1, Col3a1, Ctgf, Dcn, F2r and Fn1 in WT and Fib-3 KO CSs. Unpaired t-test, p > 0.05 = ns, p < 0.05 = *, p < 0.01 = ** and p < 0.001 = ***. Error bars represent the mean ± SD (n = 4).

Figure 10. Changes in fibrotic genes in WT and Fib-3 KO CSs following I/R injury. Relative expression of Col11a1, Col1a1, Col3a1, Ctgf, Dcn, F2r and Fn1 in WT and Fib-3 KO CSs. Two-way ANOVA with Sidak's multiple comparison test, p > 0.05 = ns, p < 0.05 = *, p < 0.01 = ** and p < 0.001 = ***. Error bars represent the mean ± SD (n = 4).