The generation of new myocytes is an essential process of in utero heart growth. Most, or all, cardiac myocytes lose their capacity for proliferation during the perinatal period through the process of terminal differentiation. An increasing number of studies focus on how experimental interventions affect cardiac myocyte growth in the fetal sheep. Nevertheless, fundamental questions about normal growth of the fetal heart remain unanswered. In this study, we determined that during the last third of gestation the hearts of fetal sheep grew primarily by four processes. 1) Myocyte proliferation contributed substantially to daily cardiac mass gain, and the number of cardiac myocytes continued to increase to term. 2) The (hitherto unrecognized) contribution to cardiac growth by the increase in myocyte size associated with the transition from mononucleation to binucleation (terminal differentiation) became considerable from approximately 115 days of gestational age (dGA) until term (145dGA). Because binucleation became the more frequent outcome of myocyte cell cycle activity after approximately 115dGA, the number of binucleated myocytes increased at the expense of the number of mononucleated myocytes. Both the interval between nuclear divisions and the duration of cell cycle activity in myocytes decreased substantially during this same period. Finally, cardiac growth was in part due to enlargement of 3) mononucleated and 4) binucleated myocytes, which grew in cross-sectional diameter but not length during the last third of gestation. These data on normal cardiac growth may enable a more detailed understanding of the consequences of experimental and pathological interventions in prenatal life.

Studies in altricial rodents attribute dramatic changes in perinatal cardiomyocyte growth, maturation, and attrition to stimuli associated with birth. Our purpose was to determine whether birth is a critical trigger controlling perinatal cardiomyocyte growth, maturation and attrition in a precocial large mammal, sheep (Ovis aries). Hearts from 0-61 d postnatal lambs were dissected or enzymatically dissociated. Cardiomyocytes were measured by micromorphometry, cell cycle activity assessed by immunohistochemistry, and nuclear number counted after DNA staining. Integration of this new data with published fetal data from our laboratory demonstrate that a newly appreciated >30% decrease in myocyte number occurred in the last 10 d of gestation (P < 0.0005) concomitant with an increase in cleaved poly (ADP-ribose) polymerase 1 (P < 0.05), indicative of apoptosis. Bisegmental linear regressions show that most changes in myocyte growth kinetics occur before birth (median = 15.2 d; P < 0.05). Right ventricular but not left ventricular cell number increases in the neonate, by 68% between birth and 60 d postnatal (P = 0.028). We conclude that in sheep few developmental changes in cardiomyocytes result from birth, excepting the different postnatal degrees of free wall hypertrophy between the ventricles. Furthermore, myocyte number is reduced in both ventricles immediately before term, but proliferation increases myocyte number in the neonatal right ventricle.-Jonker, S. S., Louey, S., Giraud, G. D., Thornburg, K. L., Faber, J. J. Timing of cardiomyocyte growth, maturation, and attrition in perinatal sheep. FASEB J. 29, 4346-4357 (2015). www.fasebj.org

Two to eight days after surgery, we determined biventricular cardiac output with the microsphere-dilution method and individual ventricular output with an electromagnetic flow sensor in 12 fetal lambs. Mean biventricular output was 462 ml.min-1.kg-1. Right ventricular output was 60%, pulmonary blood flow was 6%, blood flow in the ductus arteriosus was 54%, flow through the foramen ovale was 34%, and flow in the descending thoracic aorta above the ductus arteriosus was 12% of the biventricular cardiac output. Foramen ovale flow did not correlate with the pressure difference between the atria. The flow though the foramen ovale correlated positively with the flow in the inferior caval vein. It was concluded the shunt flow through the foramen ovale is mainly determined by the kinetic energy of the blood in the "lower atrial inflow tract." Fetal systemic somatic blood flow showed a reliable negative correlation with the oxygen tension in the fetal arterial blood. This was taken as presumptive evidence for long-term autoregulation of flow in the peripheral tissues.

Jonker SS, Faber JJ, Anderson DF, Thornburg KL, Louey S, Giraud GD. Sequential growth of fetal sheep cardiac myocytes in response to simultaneous arterial and venous hypertension. Am J Physiol Regul Integr Comp Physiol 292: R913-R919, 2007. First published October 5, 2006; doi:10.1152/ajpregu.00484.2006.-While the fetal heart grows by myocyte enlargement and proliferation, myocytes lose their capacity for proliferation in the perinatal period after terminal differentiation. The relationship between myocyte enlargement, proliferation, and terminal differentiation has not been studied under conditions of combined arterial and venous hypertension, as occurs in some clinical conditions. We hypothesize that fetal arterial and venous hypertension initially leads to cardiomyocyte proliferation, followed by myocyte enlargement. Two groups of fetal sheep received intravascular plasma infusions for 4 or 8 days (from 130 days gestation) to increase vascular pressures. Fetal hearts were arrested in diastole and dissociated. Myocyte size, terminal differentiation (%binucleation), and cell cycle activity cells as a % of mononucleated myocytes) were measured. We found that chronic plasma infusion greatly increased venous and arterial pressures. Heart (but not body) weights were ϳ30% greater in hypertensive fetuses than controls. The incidence of cell cycle activity doubled in hypertensive fetuses compared with controls. After 4 days of hypertension, myocytes were (ϳ11%) longer, but only after 8 days were they wider (ϳ12%). After 8 days, %binucleation was ϳ50% greater in hypertensive fetuses. We observed two phases of cardiomyocyte growth and maturation in response to fetal arterial and venous hypertension. In the early phase, the incidence of cell cycle activity increased and myocytes elongated. In the later phase, the incidence of cell cycle activity remained elevated, %binucleation increased, and cross sections were greater. This study highlights unique fetal adaptations of the myocardium and the importance of experimental duration when interpreting fetal cardiac growth data.hypertrophy; hyperplasia; terminal differentiation; cardiomyocyte THE FETAL HEART GROWS BY PROLIFERATION and enlargement of cardiac myocytes (23). Among mammals, proliferative growth of cardiac myocytes ceases at or soon after birth through a process known as terminal differentiation (18). Thereafter, the postnatal heart grows almost exclusively by cellular enlargement. In the fetal sheep, as in many other species, terminal differentiation is marked by the appearance of two nuclei in cardiac myocytes that were formerly mononucleated. The rate of proliferation during intrauterine life and the occurrence of terminal differentiation in the perinatal period together determine the maximum number of myocytes in the heart for life.Intrauterine conditions affect cardiac myocyte proliferation, enlargement, and terminal differentiation, but myocyte responses to abnormal conditions depend on the level of maturity of the heart. Increased systolic or diastolic hemo...

During fetal life the myocardium expands through replication of cardiomyocytes. In sheep, cardiomyocytes begin the process of becoming terminally differentiated at about 100 gestation days out of 145 days term. In this final step of development, cardiomyocytes become binucleated and stop dividing. The number of cells at birth is important in determining the number of cardiomyocytes for life. Therefore, the regulation of cardiomyocyte growth in the womb is critical to long term disease outcome. Growth factors that stimulate proliferation of fetal cardiomyocytes include angiotensin II, cortisol and insulin-like growth factor-1. Increased ventricular wall stress leads to short term increases in proliferation but longer term loss of cardiomyocyte generative capacity. Two normally circulating hormones have been identified that suppress proliferation: atrial natriuretic peptide (ANP) and tri-iodo-L-thyronine (T3). Atrial natriuretic peptide signals through the NPRA receptor that serves as a guanylate cyclase and signals through cGMP. ANP powerfully suppresses mitotic activity in cardiomyocytes in the presence of angiotensin II in culture. Addition of a cGMP analogue has the same effect as ANP. ANP suppresses both the extracellular receptor kinases and the phosphoinositol 3 kinase pathways. T3 also suppresses increased mitotic activity of stimulated cardiomyocytes but does so by increasing the cell cycle suppressant, p21, and decreasing the cell cycle activator, cyclin D1.