Figure 1.

Exposure of fetal cardiomyocytes (fCMs) to O₂ inhibits proliferation and cell cycle activity. (a) FACS analyses of E16 fCMs stained for α-actinin indicate > 95% purity when isolated and cultured under low O₂ conditions. (b) Immunofluorescence for α-actinin observed in phalloidin, DAPI, and bright-field images of E16 fCMs. The arrowhead indicates one of the few contaminating fibroblasts, which have a distinct phase appearance and are completely negative for α-actinin. (c) The fCMs (E14–E16 or E16–E18) isolated under low O₂ conditions were cultured under low or high O₂ conditions for 96 h. At the start and end of the culture, total cell numbers were counted, and the proportion of CMs and non-CMs were determined by FACS to obtain the absolute number of each cell type. n = 3–5 independent experiments. ^* P < 0.05 and ^** P < 0.01 compared to pre-culture levels. n.s. = not significant. (d,f) Immunofluorescence for Ki67 (d) and pH3 (f) observed in α-actinin and DAPI of low O₂-isolated fCMs cultured under low or high O₂ conditions. Arrows denote Ki67- (d) and pH3- (f) positive fCMs. (e,g) Proportions of Ki67- (e) and pH3- (g) positive CMs in (d) and (f), respectively. n = 5 independent experiments. ^* P < 0.05. (h) Rb protein expressions in low O₂-isolated fCMs (E16–E17) cultured under low or high O₂ conditions. β-tubulin was used as a loading control. n = 5 independent experiments. ^* P < 0.05. Error bars = SEM. Scale bars = 20 µm in (b) and 50 µm in (d,f).