Placental insufficiency disrupts cardiomyocyte ploidy and cell cycle fate in growth-restricted fetal sheep

Cardiomyocytes undergo proliferation, differentiation, and hypertrophy during fetal development. Current techniques struggle to distinguish cardiomyocyte proliferation from alternative cell fates. In this study, we combined flow cytometry measures of cardiomyocyte ploidy (DAPI) and in vivo DNA replication (EdU) over a 24-hour period to evaluate the trajectories of cardiomyocytes from normally-growing control fetal sheep and fetuses affected by placental insufficiency and fetal growth restriction (FGR) at 0.9 gestation. We categorized ~100,000 cardiomyocytes from the left and right ventricles (LV, RV) of each animal as proliferating (2C EdU + ), differentiated (4C EdU - ), or endoreplicating and polyploid (6C+ EdU + ). Compared to controls, FGR hearts had 25-50% fewer cardiomyocytes that replicated DNA (EdU + ) (LV: P=0.02, RV: P=0.002). The fraction of proliferating cardiomyocytes, indicated by the population of newly synthesized 2C EdU + daughter cells, was ~20% lower in FGR fetuses (LV: P=0.006, RV: P=0.02). Instead, the percentage of endoreplicating cardiomyocytes (6C+ EdU + ) in FGR hearts was double that of controls (LV: P=0.004, RV: P=0.002). While total EdU + was not a strong predictor of cardiac growth, LV and RV mass correlated positively with the percentage of 2C EdU + cardiomyocytes and negatively with 6C+ EdU + cardiomyocytes across all fetuses. LV mass also correlated positively with the percentage of differentiated cardiomyocytes (4C EdU - ), which was lower in FGR hearts compared to controls ( P=0.008). This study is the first to characterize cardiomyocyte fate following DNA replication in fetal sheep. Our findings suggest that FGR cardiomyocytes differentially prioritize their cycling capacity in favor of polyploidization instead of proliferation.