Cardiomyocyte enlargement, proliferation and maturation during chronic fetal anaemia in sheep

Abstract

Chronic anaemia increases the workload of the growing fetal heart, leading to cardiac enlargement. To determine which cellular process increases cardiac mass, we measured cardiomyocyte sizes, binucleation as an index of terminal differentiation, and tissue volume fractions in hearts from control and anaemic fetal sheep. Fourteen chronically catheterized fetal sheep at 129 days gestation had blood withdrawn for 9 days to cause severe anaemia; 14 control fetuses were of similar age. At postmortem examination, hearts were either enzymatically dissociated or fixed for morphometric analysis. Daily isovolumetric haemorrhage reduced fetal haematocrit from a baseline value of 35% to 15% on the final day (P < 0.001). At the study conclusion, anaemic fetuses had lower arterial pressures than control fetuses (P < 0.05). Heart weights were increased by 39% in anaemic fetuses compared with control hearts (P < 0.0001), although the groups had similar body weights; the heart weight difference was not due to increased ventricular wall water content or disproportionate non-myocyte tissue expansion. Cardiomyocytes from anaemic fetuses tended to be larger than those of control fetuses. There were no statistically significant differences between groups in the cardiomyocyte cell cycle activity. The degree of terminal differentiation was greater in the right ventricle of anaemic compared with control fetuses by 8% (P < 0.05). Anaemia substantially increased heart weight in fetal sheep. The volume proportions of connective and vascular tissue were unchanged. Cardiomyocyte mass expanded by a balanced combination of cellular enlargement, increased terminal differentiation and accelerated proliferation.

Figure 1

Oxygen content decreased most rapidly during the first 5 days of bleeding. Solid circles = control fetuses, open circles = anemic fetuses (n = 12 each group). Data are presented as means ± SEM.

Figure 2

(A) Percent binucleation and (B) cell cycle activity as a percent of mononucleated myocytes (as measured by Ki67 positivity) in the hearts of anemic and control fetuses. Solid bars = control fetuses (n = 9), open bars = anemic fetuses (percent binucleation: n = 9; cell cycle activity n = 8). Data are presented as means ± SEM. *Mean significantly (p < 0.05) different from ipsilateral control.

Figure 3

Freewall myocardial volume is composed of the total summed volumes of mononucleated and binucleated cardiomyocytes. The products of mononucleated myocyte number and volume are shown in white bars, while those of binucleated myocytes are shown in black bars. Data are presented as means ± SEM. Means significantly (p<0.01) different from control fetuses for *mononucleated and binucleated cardiomyocytes combined, and †binucleated cardiomyocytes.