Thymosin β4 and prothymosin α promote cardiac regeneration post-ischaemic injury in mice

Figure 1. Gene profile of cardiomyocytes expressing cell-cycle markers. (A) Experimental workflow to identify proliferating cardiomyocytes. (B) Expression level of the selected proliferation markers in the 16 identified cardiomyocytes. (C) Top 14 enriched genes expressed in sixteen identified cardiomyocytes, compared to all other cardiomyocytes. (D) Validation strategies. X = indicates the expression of a selected factor in the individual cell.

Figure 2. Proliferative potential of different combinations of factors delivered to NRCMs. (A) Experimental design. (B) mRNA expression level of eGFP (Ct values) to assess the infection efficiency in cultured NRCM. (C) Representative images (two pictures per condition) of NRCM infected with the indicated combinations (n = 4, ±100 cells per condition). Cells were stained with ACTN2, EdU and DAPI. (D) Quantification of the percentage of EdU-positive cardiomyocytes. (E) List of the top combinations selected for follow-up analysis. Scale bar represents 50 mm. Data are shown as the mean ± SEM. *P < 0.05 and **P < 0.01 compared to control using unpaired, two-tailed Student’s t-test or one-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 3. AAV9-mediated gene delivery of combinations 8 and 11 induces cardiomyocyte proliferation in vivo. (A) Experimental design. (B) Quantification of total proliferation in the heart based on the percentage of EdU positive nuclei from mice treated with the indicated combinations (n = 4, 750–1000 cardiomyocytes from 5 sections per mouse). (C) Immunofluorescent staining for EdU, WGA, ACTN2 and DAPI of hearts treated with the indicated combinations and zoomed-in magnifications of EdU positive cardiomyocytes. (D) Quantification of the percentage of EdU-positive cardiomyocytes. (E-I) mRNA expression levels of (E) Ccnd1, (F) Cdk4, (G) Cdk6, (H) Pcna, and (I) Mki67 on heart tissue from mice treated with the indicated combinations. Scale bar represents 50 mm. Data are shown as the mean ± SEM. *P < 0.05 and **P < 0.01 compared to control using one-way ANOVA followed by Dunnett’s multiple comparisons test.

Figure 4. Therapeutic AAV9 delivery of combination 11 improves cardiac function and promotes clonal expansion of cardiomyocytes post-ischaemic injury. (A) Experimental design. (B–D) Quantification of (B) EF and (C) IVRT and (D) LVPWs from AAV9-control and AAV9-C8 injected mice post-surgery (n = 6 sham, n = 15 IR). (E–G) Quantification of (E) EF (F) IVRT and (G) LVPWs from AAV9-control and AAV9-C11 injected mice post-surgery. (H) Experimental design. (I) Representative images of cardiac tissue (border zone) from aMHC-MerCreMer-Confetti mice injected with combination 11 (C11) post-injury (n = 6). (J–K) Cardiomyocyte clonal analysis based on quantification of adjacent cardiomyocytes with the same colour considered to arise from the proliferation of single-labeled cells (mononucleated cardiomyocytes (monochromatic cardiomyocytes)). Data are shown as mean ± SEM. *P< 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 using one-way ANOVA followed by Sidak’s multiple comparisons test (for B–G) and Dunnett’s multiple comparisons test (for J–K). Scale bar represents 100 mm. IC = intracardiac injection, IR = ischaemia/reperfusion.