Chronic hypoxemia in late gestation decreases cardiomyocyte number but does not change expression of hypoxia-responsive genes

Abstract

Background: Placental insufficiency is the leading cause of intrauterine growth restriction in the developed world and results in chronic hypoxemia in the fetus. Oxygen is essential for fetal heart development, but a hypoxemic environment in utero can permanently alter development of cardiomyocytes. The present study aimed to investigate the effect of placental restriction and chronic hypoxemia on total number of cardiomyocytes, cardiomyocyte apoptosis, total length of coronary capillaries, and expression of genes regulated by hypoxia.

Methods and results: We induced experimental placental restriction from conception, which resulted in fetal growth restriction and chronic hypoxemia. Fetal hearts in the placental restriction group had fewer cardiomyocytes, but interestingly, there was no difference in the percentage of apoptotic cardiomyocytes; the abundance of the transcription factor that mediates hypoxia-induced apoptosis, p53; or expression of apoptotic genes Bax and Bcl2. Likewise, there was no difference in the abundance of autophagy regulator beclin 1 or expression of autophagic genes BECN1, BNIP3, LAMP1, and MAP1LC3B. Furthermore, fetuses exposed to normoxemia (control) or chronic hypoxemia (placental restriction) had similar mRNA expression of a suite of hypoxia-inducible factor target genes, which are essential for angiogenesis (VEGF, Flt1, Ang1, Ang2, and Tie2), vasodilation (iNOS and Adm), and glycolysis (GLUT1 and GLUT3). In addition, there was no change in the expression of PKC-ε, a cardioprotective gene with transcription regulated by hypoxia in a manner independent of hypoxia-inducible factors. There was an increased capillary length density but no difference in the total length of capillaries in the hearts of the chronically hypoxemic fetuses.

Conclusion: The lack of upregulation of hypoxia target genes in response to chronic hypoxemia in the fetal heart in late gestation may be due to a decrease in the number of cardiomyocytes (decreased oxygen demand) and the maintenance of the total length of capillaries. Consequently, these adaptive responses in the fetal heart may maintain a normal oxygen tension within the cardiomyocyte of the chronically hypoxemic fetus in late gestation.

Keywords: angiogenesis; apoptosis; hypoxia; myocytes; pregnancy.

Figure 1.

Placental restriction (PR) resulting in chronic hypoxemia reduced the total number of cardiomyocytes (A) and binucleated cardiomyocytes (C) but not mononucleated cardiomyocytes (B) in the right ventricle. The total number of cardiomyocytes is positively correlated with fetal weight (D). *P<0.05; Control, ○; PR, ●.

Figure 2.

Placental restriction (PR) resulting in chronic hypoxemia did not alter the percentage of apoptotic cardiomyocytes (A) or the mRNA expression of pro‐apoptotic gene Bax (B) or antiapoptotic gene Bcl2 (C) in the right ventricle. PR did not affect the protein abundance of hypoxia‐mediated apoptosis regulator p53 (D) but resulted in decreased mRNA expression of both pro‐apoptotic gene Bax (E) and antiapoptotic gene Bcl2 (F) in the left ventricle. Open circle represents an outlier, defined as being >1.5×IQR; *P<0.05. MNE indicates mean normalized expression; TUNEL, terminal deoxynucleotidyl transferase dUTP nick‐end labeling.

Figure 3.

Placental restriction (PR; A and F) resulting in chronic hypoxemia increased capillary length density in the right ventricle compared with controls (A and E); however, there was a similar total length of capillaries (B). In the control right ventricle, there was a significant positive correlation between the total number of cardiomyocytes and the total length of capillaries; however, this correlation was not present in the PR group (C). Note, analysis of combined data from the control and PR groups demonstrated a significant correlation between the total length of capillaries and cardiomyocyte number (r=0.620, P=0.042); however, simple/linear regression analysis failed to reach significance. There is an increase in the length of capillaries per cardiomyocyte in the PR group compared with the control group (D; presented as arbitrary units (au) due to the 2 analyses being performed in sections embedded in different compounds; control, n=7; PR, n=4; these numbers are reduced because not every animal had both components analyzed). Coronary capillaries were identified with immunohistochemistry for α‐smooth muscle actin in the pericytes that surround the capillaries (brown) and counterstained with Mayer's hematoxylin. An open circle represents an outlier, defined as being >1.5×IQR; *P<0.05.

Figure 4.

PR resulting in chronic hypoxemia does not change the protein abundance of PHD1 (A) but increases the protein abundance of PHD2 (B) in the left ventricle. Treatment groups were alternated across the Western blot to minimize transfer bias; *P<0.05. PHD indicates prolyl hydroxylase domain; PR, placental restriction.