Suitable protocols for reproducing active proliferation in culture sufficient for mechanistic examinations are lacking in mammalian fetal cardiomyocytes (fCMs). Because oxygen tension is low (2-3%) in mammalian fetuses before birth, we assume that the exposure of cells to ambient air (21% oxygen) during conventional isolation inhibits the proliferative activity of isolated fCMs. We therefore established an isolation protocol performed under strict low oxygen conditions to mimic the intrauterine environment. This protocol greatly increased mouse fCM division in culture, which was directly observed by time-lapse imaging, to the highest level thus far reported. Even short-term oxygen exposure during isolation markedly inhibited cell division by >70% and repressed cell cycle-promoting gene expression, indicating the absolute requirement of strict low oxygen maintenance in fCMs. Using genome-wide screening, we identified Fam64a as a low oxygen-induced molecule essential for fetal cardiomyocyte proliferation. These data could direct future regenerative therapies using Fam64a in adult hearts.
