Angiotensin II–induced cardiovascular load regulates cardiac remodeling and related gene expression in late-gestation fetal sheep

Abstract

Background Angiotensin II (ANG II) stimulates fetal heart growth, though little is known regarding changes in cardiomyocyte endowment or the molecular pathways mediating the response. We measured cardiomyocyte proliferation and morphology in ANG II treated fetal sheep and assessed transcriptional pathway responses in ANG II and losartan (an ANG II type 1 receptor antagonist) treated fetuses. Methods In twin gestation pregnant sheep, one fetus received ANG II (50 μg/kg/min iv) or losartan (20 mg/kg/d iv) for 7 days; non-instrumented twins served as controls. Results ANG II produced increases in heart mass, cardiomyocyte area (left ventricle (LV) and right ventricle mononucleated and LV binucleated cells) and the percentage of Ki-67-positive mononucleated cells in the LV (all p< 0.05). ANG II and losartan produced generally opposing changes in gene expression, affecting an estimated 55% of the represented transcriptome. The most prominent significantly effected biological pathways included those involved in cytoskeletal remodeling and cell cycle activity. Conclusion ANG II produces an increase in fetal cardiac mass via cardiomyocyte hypertrophy and likely hyperplasia, involving transcriptional responses in cytoskeletal remodeling and cell cycle pathways.

Endocrine and other physiologic modulators of perinatal cardiomyocyte endowment

“…AII causes near-term fetal sheep cardiomyocytes to proliferate in culture, contrary to its hypertrophic effect in cultured neonatal rat cardiomyocytes (O’Tierney et al 2010; Sundgren et al 2003). In contrast, AII infusion in utero increases ovine heart growth via myocyte hypertrophy and maturation (Norris et al 2014; Sandgren et al 2015; Segar et al 2001). Atrial natriuretic peptide (ANP) inhibits AII-stimulated fetal cardiomyocyte proliferation in culture (O’Tierney et al 2010); increased arterial pressure resulting from in utero AII infusion may simultaneously inhibit proliferation by stimulating ANP release while stimulating cellular enlargement and terminal differentiation via increased wall stress.…”

ANG II modulation of cardiac growth and remodeling in immature fetal sheep

“…ANG II REGULATES FETAL HEART GROWTH significantly increased fetal mean blood pressure by 15-20 mmHg, resulted in increased cardiac mass via cardiomyocyte hypertrophy and proliferation (26,36). However, in these studies, we were unable to differentiate the mechanisms by which ANG II affects cardiomyocytes.…”

“…For determination of ploidy, cells were stained with hematoxylin and eosin, and no fewer than 300 myocytes from each ventricle of each fetus were analyzed at ϫ40 magnification on a light microscope (Zeiss Axiophot, Bartels and Stout, Bellevue, WA) to determine the number of nuclei per cardiomyocyte. For cell morphology, long-axis (length) and maximal cross-sectional diameter (width) dimensions and area of cardiomyocytes were measured, as previously described (26). Fixed myocytes were prepared in a wet mount with methylene blue and were selected for measurement, according to a random, nonrepeating, and unbiased method employing a counting frame.…”

“…We recently utilized gene expression arrays to explore changes in the late-gestation fetal cardiac transcriptome associated with ANG II-induced increased cardiac mass (26). Chief among the biological pathways identified as highly impacted by fetal RAS modulation are those involved in cytoskeletal remodeling, transcriptional regulation, and cell cycle activity.…”

“…In vitro, ANG II stimulates fetal cardiomyocyte proliferation, but not hypertrophy (39). In vivo, infusion of ANG II results in late-gestation fetal cardiomyocyte proliferation and hypertrophy; however, whether this is a direct or indirect effect, resulting from increased blood pressure and mechanical load is not known (26). Therefore, we sought to determine whether ANG II stimulates heart growth and cardiomyocyte hyperplasia and/or hypertrophy in the immature fetal heart independent of the effects of cardiac load.…”