Oxytocin in cardiac ontogeny

Abstract

Previous studies demonstrated the presence of oxytocin (OT) and oxytocin receptors (OTRs) in the heart. The present work provides results supporting a potential role of OT in cardiomyogenesis. Here, we show a maximal OT and OTR protein level in the developing rat heart at day 21 of gestation and postnatal days 1-4, when cardiac myocytes are at a stage of intense hyperplasia. Between postnatal days 1 and 66, OT decreased linearly in all heart chambers (4.1- to 6.6-fold). Correspondingly, immunocytochemistry demonstrated that OTRs, which were eminent in postnatal cardiomyocytes, declined with age to low levels in adults. Interestingly, in coronary vasculature, OTRs developed in endothelial cells at postnatal days 12 and 22 and achieved a plateau in adult rats. These findings suggest that OT can be involved in developmental formation of the coronary vessels. In vivo, the OT/OTR system in the fetal heart was sensitive to the actions of retinoic acid (RA), recognized as a major cardiac morphogen. RA treatment produced a significant increase (2- to 3-fold) both in the OT concentration and in the OT mRNA levels. Ex vivo, an OT antagonist inhibited RA-mediated cardiomyocyte differentiation of P19 embryonic stem cells. The decline of cardiac OT expression from infancy to adulthood of the rat and changes in cell types expressing OTR indicate a dynamic regulation of the OT system in the heart rather than constitutive expression. The results support the hypothesis that RA induces cardiomyogenesis by activation of the cardiac OT system.

Fig. 1.

RA activates OT systems in the fetal heart (E21). OT concentration was measured by RIA (A, n = 11; ^*, P < 0.05) and OT mRNA by semiquantitative RT-PCR (B, n = 8; ^*, P < 0.05). (C) ¹²⁵I-OTA binding to the fetal sections demonstrated by autoradiography. The representative competition curve of ¹²⁵I-OTA binding to the fetal heart sections by unlabeled OTA is shown. (D) Retinal dehydrogenase type 1 mRNA detected by Northern blotting in the fetal heart (lane 1) and kidney (lane 2).

Fig. 2.

Changes in OT concentration during rat postnatal development measured by RIA in the heart chambers (A, atria; B, ventricles; n = 6-12; ^*, P < 0.05, indicating increase vs. preceding age group) and hypothalamus (C; n = 6-8; ^*, P < 0.05).

Fig. 3.

Atrial OT and OTR during rat maturation. (A) RT-PCR for OT and OTR mRNA in the left atria of newborn (PD 4) and adult (PD 66) rats, n = 6; ^*, P < 0.05. (B) Corresponding Western blot analysis of OTR expression in the left atrium, n = 4; ^*, P < 0.05.

Fig. 4.

ICC staining. (A-A″) OTR ICC staining (arrows) in adult rat fallopian tubes at low (A) and higher (A′ and A″) magnifications. Heavy arrows, smooth muscles; long thin arrows, epithelial cells; small arrows, stroma macrophages. OTR ICC sites (arrows) in atrial cardiomyocytes at low (B) and high (B′) magnification of a 6-day-old rat heart. For comparison, ANP immunoreactivity (IR-ANP) is shown at low (C) and higher (C′) magnification of adjacent section. Adult rat atrium OTR ICC sites (arrows) are shown in D, and coronary artery is in E. Long arrows, endothelial cells; Adv, tunica adventicia; En, endothelial cells; Med, tunica media. (×40 in A-C; ×405 in A′, A″, B′, and C′; ×160 in D and E. Bar = 100 μm in A-E; bar = 10 μm in A′, A″, B′, and C′.)

Fig. 5.

OTR mRNA expression in human adult heart (AH) and fetal heart (FH) detected by RT-PCR. The studies were performed in cDNA samples from human hearts (Human Cardiovascular System MTC Panel no. 636749, Clontech). (Lower) Representative PhoshorImager-scanned bands of PCR products amplified with specific primers for OTR and GAPDH used as internal standard. (Upper) The mean OTR value from three independent amplifications after adjusting the GAPDH value. A, aorta; RT, right atrium; LA, left atrium; RV, right ventricle; LV, left ventricle; IS, interventricular septum; AN, atrioventricular node; AP, apex of the heart; AD, right auricle; AS, left auricle.

Fig. 6.

Cardiomyogenic effect of OT and RA in P19 cells is suppressed by OTA. (A) Time course of appearance of beating cell colonies upon treatment with OT and RA in presence and absence of OTA. The number of wells containing beating cell colonies was calculated in a 24-well tissue culture plate at 2-day intervals. The results are means of three independent differentiation experiments. (B) RT-PCR analysis of OTR gene transcript in undifferentiated and induced cultures. Cell aggregates were exposed to OT (10^-7 M) or RA (10^-7 M) in the absence or presence of OTA (10^-7 M) from day 0 to day 4, and RNA was extracted at day 14 of the differentiation protocol. OTR transcript also was evaluated in undifferentiated cells grown in monolayers (NI). Levels of OTR mRNA were adjusted by dividing by corresponding GAPDH mRNA and then expressed as the percentage of the NI value. Results are the means ± SEM of five independent studies (^*, P < 0.05). (C) Monitoring of P19 cell differentiation into cardiomyocytes by using selective vital staining with rhodamine¹²³ in noninduced and OT- and RA-induced cultures. On day 8, the cells were stained for 45 min with rhodamine¹²³ (1 μg/ml), washed, and again cultured without dye for 48 h. Then, the retained dye was fluorimetrically quantified for each well, and the results are reported as the means ± SEM of 24 determinations (^*, P < 0.05).