Endothelin-1 promotes cardiomyocyte terminal differentiation in the developing heart via heightened DNA methylation

Abstract

Aims: Hypoxia is a major stress on fetal development and leads to induction of endothelin-1 (ET-1) expression. We tested the hypothesis that ET-1 stimulates the terminal differentiation of cardiomyocytes from mononucleate to binucleate in the developing heart.

Methods and results: Hypoxia (10.5% O2) treatment of pregnant rats from day 15 to day 21 resulted in a significant increase in prepro-ET-1 mRNA expression in fetal hearts. ET-1 ex vivo treatment of fetal rat cardiomyocytes increased percent binucleate cells and decreased Ki-67 expression, a marker for proliferation, under both control and hypoxic conditions. Hypoxia alone decreased Ki-67 expression and in conjunction with ET-1 treatment decreased cardiomyocyte size. PD145065, a non-selective ET-receptor antagonist, blocked the changes in binucleation and proliferation caused by ET-1. DNA methylation in fetal cardiomyocytes was significantly increased with ET-1 treatment, which was blocked by 5-aza-2'-deoxycytidine, a DNA methylation inhibitor. In addition, 5-aza-2'-deoxycytidine treatment abrogated the increase in binucleation and decrease in proliferation induced by ET-1.

Conclusions: Hypoxic stress and synthesis of ET-1 increases DNA methylation and promotes terminal differentiation of cardiomyocytes in the developing heart. This premature exit of the cell cycle may lead to a reduced cardiomyocyte endowment in the heart and have a negative impact on cardiac function.

Keywords: Endothelin-1; Epigenetic; Fetal development; Heart; Hypoxia.

Figure 1

Effect of hypoxia on prepro-ET1 mRNA in the fetal heart. Hearts were isolated from near-term fetuses of pregnant rats treated with control or hypoxia. mRNA abundance of prepro-ET-1 was determined by real-time RT-PCR. Data are means ± SEM. * P < 0.05, hypoxia vs. control. n = 7-8

Figure 2

Effect of ET-1 on binucleation and proliferation of fetal cardiomyocytes. Cardiomyocytes isolated from fetal hearts were treated with ET-1 (10 nM) under normoxic control (21% O₂) or hypoxic (1% O₂) conditions for 24 h. A. Morphology of mononucleate and binucleate fetal cardiomyocytes. B. Binucleation result. C. Proliferation result. Data are means ± SEM. * P < 0.05, +ET-1 vs. -ET-1; † P < 0.05, hypoxia vs. control. n = 5

Figure 3

Effect of ET-1 on fetal cardiomyocyte size. Cardiomyocytes isolated from fetal hearts were treated with ET-1 (10 nM) under normoxic control (21% O₂) or hypoxic (1% O₂) conditions for 24 h. Data are means ± SEM. * P < 0.05, +ET-1 vs. -ET-1. n = 7-10

Figure 4

PD145065 abrogates ET-1-mediated effects on binucleation and proliferation of fetal cardiomyocytes. Cardiomyocytes isolated from fetal hearts were treated with ET-1 (10 nM) for 24 h in the absence or presence of PD145065 (10 nM). A. Binucleation result. B. Proliferation result. * P < 0.05, ET-1 vs. control. n = 5

Figure 5

5-Aza-2'-deoxycytidine blocks ET-1-increased DNA methylation in fetal cardiomyocytes. Cardiomyocytes isolated from fetal hearts were treated with ET-1 (10 nM) for 24 h in the absence or presence of 5-aza-2'-deoxycytidine (5-Aza, 10 µM). * P < 0.05, ET-1 vs. control. n = 5

Figure 6

5-Aza-2'-deoxycytidine abrogates ET-1-mediated effects on binucleation and proliferation of fetal cardiomyocytes. Cardiomyocytes isolated from fetal hearts were treated with ET-1 (10 nM) for 24 h in the absence or presence of 5-aza-2'-deoxycytidine (5-Aza, 10 µM). A. Binucleation result. B. Proliferation result. * P < 0.05, ET-1 vs. control. n = 5