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. Author manuscript; available in PMC: 2016 Jul 14.
Published in final edited form as: Circulation. 2015 May 20;132(2):109–121. doi: 10.1161/CIRCULATIONAHA.114.011490

Figure 4.

Figure 4

Embryonic CM proliferation is involved in myocardial recovery. (A) Schematic diagram of single-cell isolation and culture of E9.5 cardiac cells. For (B-J): experimental variables are represented as follows: [group: # of mice examined, mean percent ablation ± SD]. For (B-F): [control: n=9; CPC ablation: n=6, 45 ± 11%; CM ablation: n=6, 50 ± 12%]. (B) Percentage of single-cell derived colonies classified as cardiomyocytes, endocardial/endothelial cells, or other cells from dissociated E9.5 embryo hearts. (C-E) Histological analysis of single-cell derived colonies from E9.5 hearts. (C) Sarcomeric α-actinin+ cardiomyocytes showing well-organized Z-lines and sarcomeric structure. (D) CD31+ endocardial/endothelial cells. (E) Non-cardiomyocyte/non-endothelial cell lacking significant sarcomeric α-actinin or CD31 expression. (F) Box-and-whisker plot demonstrating median cell number and interquartile range among wells classified as containing cardiomyocyte colonies. Whiskers span the 10-90% range of values. Statistical comparisons were performed using the Kruskal-Wallis test with Dunn's correction for multiple comparisons. For (G-J): [control: n=4; CPC ablation: n=4, 41 ± 9%; CM ablation: n=4, 53 ± 12%]. Immunofluorescence confocal microscopy with 1 mm z-stack imaging for proliferative cardiomyocytes and endocardial cells in CPC-ablated (G and H) or CM-ablated (I and J) hearts at E8.5 or E9.5, respectively. The percentage of phospho-histone H3+ (pH3) (G and I) or Ki-67+ (H and J) cardiomyocytes in control and ablated hearts (filled arrows) was defined by co-localization with a DAPI+ nucleus that is circumscribed in all dimensions by a troponin T+ (cTnT) cell. Similarly, the percentage of phospho-histone H3+ or Ki-67+ endocardial cells (thin arrows) was defined by co-localization with a DAPI+ nucleus that is circumscribed in all dimensions with a CD31+ cell. Each symbol in the dot plot represents the fraction of cTnT+ or CD31+ cells staining positive for pH3 or Ki-67 in a single cardiac histology section; each symbol shape represents an independent mouse; a horizontal bar indicates the mean value for each group. 100-600 cTnT+ cells and 50-300 CD31+ cells were counted per section. Statistical comparisons were performed using a likelihood ratio test on a linear mixed-effects model. NS, non-significant. Scale bars: 100 μm for (C-E); 50 μm for (G-J).