Elabela/Toddler Is an Endogenous Agonist of the Apelin APJ Receptor in the Adult Cardiovascular System, and Exogenous Administration of the Peptide Compensates for the Downregulation of Its Expression in Pulmonary Arterial Hypertension

Abstract

Background: Elabela/toddler (ELA) is a critical cardiac developmental peptide that acts through the G-protein-coupled apelin receptor, despite lack of sequence similarity to the established ligand apelin. Our aim was to investigate the receptor pharmacology, expression pattern, and in vivo function of ELA peptides in the adult cardiovascular system, to seek evidence for alteration in pulmonary arterial hypertension (PAH) in which apelin signaling is downregulated, and to demonstrate attenuation of PAH severity with exogenous administration of ELA in a rat model.

Methods: In silico docking analysis, competition binding experiments, and downstream assays were used to characterize ELA receptor binding in human heart and signaling in cells expressing the apelin receptor. ELA expression in human cardiovascular tissues and plasma was determined using real-time quantitative polymerase chain reaction, dual-labeling immunofluorescent staining, and immunoassays. Acute cardiac effects of ELA-32 and [Pyr¹]apelin-13 were assessed by MRI and cardiac catheterization in anesthetized rats. Cardiopulmonary human and rat tissues from PAH patients and monocrotaline- and Sugen/hypoxia-exposed rats were used to show changes in ELA expression in PAH. The effect of ELA treatment on cardiopulmonary remodeling in PAH was investigated in the monocrotaline rat model.

Results: ELA competed for binding of apelin in human heart with overlap for the 2 peptides indicated by in silico modeling. ELA activated G-protein- and β-arrestin-dependent pathways. We detected ELA expression in human vascular endothelium and plasma. Comparable to apelin, ELA increased cardiac contractility, ejection fraction, and cardiac output and elicited vasodilatation in rat in vivo. ELA expression was reduced in cardiopulmonary tissues from PAH patients and PAH rat models, respectively. ELA treatment significantly attenuated elevation of right ventricular systolic pressure and right ventricular hypertrophy and pulmonary vascular remodeling in monocrotaline-exposed rats.

Conclusions: These results show that ELA is an endogenous agonist of the human apelin receptor, exhibits a cardiovascular profile comparable to apelin, and is downregulated in human disease and rodent PAH models, and exogenous peptide can reduce the severity of cardiopulmonary remodeling and function in PAH in rats. This study provides additional proof of principle that an apelin receptor agonist may be of therapeutic use in PAH in humans.

Keywords: Elabela/Toddler; apelin; cardiopulmonary; pulmonary hypertension; receptors, G-protein-coupled.

Figure 1.

ELA peptides bind to the human apelin receptor. A, Amino acid sequences of ELA-32, ELA-21, ELA-11, and [Pyr¹]apelin-13. Disulfide bridges are yellow lines, hydrophobic amino acids are shown in green, uncharged polar amino acids in pink, basic amino acids in blue with pyroglutamate in red. B, Docking of ELA-11 with the apelin receptor. C, Apelin-13 (multicolored atoms) and ELA-11 (green) docking showing overlap in the binding site. D, Ligand interaction diagram showing key close contacts between ELA-11 and the apelin receptor model. E, Predicted interactions of ELA-11 with the apelin receptor determined by docking analysis. Amino acids in the ELA-11 sequence are color coded to match their predicted sites of interaction on the receptor sequence. ELA indicates Elabela/toddler.

Figure 2.

Competition binding curves for ELA peptides in human heart. A, ELA-32, ELA-21, ELA-11, and [Pyr¹]apelin-13 in human left ventricle. B, ELA-21 in normal right ventricle, PAH right ventricle, normal left ventricle, and PAH left ventricle. Values are mean±SEM, n=3. ELA indicates Elabela/toddler; PAH, pulmonary arterial hypertension; and SEM, standard error of the mean.

Figure 3.

Concentration-response curves in cell-based receptor pharmacology assays. A, Inhibition of forskolin-induced cAMP accumulation. B, Stimulation of β-arrestin recruitment. C, Induction of receptor internalization. [Pyr¹]apelin-13, ELA-32, ELA-21, and ELA-11. Antagonism of [Pyr¹]apelin-13 (D) and ELA-32 (E) by 30 μmol/L ML221 in the β-arrestin assay. Concentration-response curves to ELA-14 and cyclic ELA-11 in cAMP (F) and β-arrestin (G) assays. Values are mean±SEM, n=3 to 5 assays, 2 to 6 replicates per assay. ELA indicates Elabela/toddler; and SEM, standard error of the mean.

Figure 4.

Expression of APELA transcript and ELA peptide in human cardiovascular tissues. A, Expression of APELA mRNA in human blood vessels determined by RT-qPCR. B through F, Representative overlay confocal photomicrographs of immunofluorescence staining of human pulmonary artery endothelial cell (PAEC) (B) and human cardiovascular tissue (C through F). Photomicrographs show ELA-like immunoreactivity in green and the endothelial marker vWF in red. If not indicated, scale bars=75 μm. CA, coronary artery (n=11); MA, mammary artery (n=6); RA, radial artery (n=9); PA, pulmonary artery (n=4); UV, umbilical vein (n=6); AO, aorta (n-6); SV, saphenous vein (n=8); LV, left ventricle (n=8); lung (n=6). G, Levels of ELA and apelin in human plasma (n=25). H, Correlation between plasma concentrations of ELA and apelin. *P<0.05 in comparison with ELA. ELA indicates Elabela/toddler; RT-qPCR, real-time quantitative polymerase chain reaction; and vWF, von Willebrand factor.

Figure 5.

Cardiac effects of ELA and apelin in vivo by MRI in the rat. Representative snapshots of midventricular transverse sections of the heart at end-diastolic and end-systolic points during the 10-minute MRI scan showing the effects of ELA-32 (150 nmol) (A) and [Pyr¹]apelin-13 (650 nmol) (B). Red bars are drawn to show the approximate diameter of the left ventricle. Dose-dependent increase in left (C) and right (D) ventricular ejection fraction of the heart in response to ELA-32 (red bars, 20 and 150 nmol, n=8) and [Pyr¹]apelin-13 (black bars, 50 and 650 nmol, n=5). *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001 in comparison with saline control (n=6). ELA indicates Elabela/toddler; LVEF, left ventricular ejection fraction; ns, not significantly different from saline control; and RVEF, right ventricular ejection fraction.

Figure 6.

The in vivo effects of ELA-32 and [Pyr¹]apelin-13 on the left ventricle of rat heart measured by catheterization. Compared to saline controls (open bars, n=6), ELA-32 (red bars, n=10), and [Pyr¹]apelin-13 (black bars, n=8) caused a significant change in cardiac contractility (dP/dt_MAX) (A), cardiac output (CO) (B), stroke volume (SV) (C), and LV systolic pressure (LVSP) (D). *P<0.05. **P<0.01. ***P<0.001. ****P<0.0001 in comparison with saline control. ELA indicates Elabela/toddler; LV, left ventricular; RVU, relative volume unit; and ns, not significantly different from saline control.

Figure 7.

Altered levels of ELA expression in PAH. A, Number of blood vessels positive and negative for ELA staining in control (n=4) and PAH (n=4) human lung sections (****P≤0.0001). B, APELA downregulation in human PAH (n=4) in comparison with normal (n=4) lung (**P≤0.01). Apela, mRNA downregulation in the right ventricle of Sugen/hypoxia (n=6) (C) and MCT exposed (n=5) (D) rats (*P≤0.05, **P≤0.01, respectively, in comparison with saline (n=7 and 5, respectively). Aplnr (E) and Apln mRNA (F) expression in right ventricle of saline (n=5) and MCT exposed (n=5) rats (*P≤0.05, in comparison with saline. ELA indicates Elabela/toddler; MCT, monocrotaline; ns, not significantly different from saline control; and PAH, pulmonary arterial hypertension.

Figure 8.

Attenuation of MCT-induced PAH by ELA-32 in rats. MCT exposure (black bar, n=9) caused an increase in right ventricular systolic pressure (RVSP) (A) and right ventricular hypertrophy (B) measured as RV/(LV+S) (Fulton index) (both ****P≤0.0001) in comparison with saline control (open bars, n=8). ELA-32 administration (red bars, n=9) significantly reduced MCT-induced RVSP and hypertrophy (****P≤0.0001 and ****P≤0.0001, respectively). ELA-32 alone (gray bars, n=9) had no effect and was not different from saline control (open bars, n=8). In tissues from these animals, MCT increased the proportion of fully muscularized vessels in rat lung (C) and wall thickness of larger pulmonary arterioles (D) in comparison with saline (both ****P≤0.0001), and these changes were attenuated by ELA-32 (**P≤0.01 and ****P≤0.0001, respectively). Immunohistological visualization of remodeling of pulmonary arterioles using α-smooth muscle actin (brown, E through H) and van Giesen stain (I through L) in sections of lung from MCT (E, I), MCT-ELA (F, J), ELA alone (G, K), and saline control rats (H, L) (scale bars=75μm). MCT-induced RV hypertrophy was indicated by an increase in cardiomyocyte area indicated by WGA staining (M), a reduction in cardiomyocyte number/area (N), and an increase in GATA4-positive nuclei/area compared to saline control (O) (****P≤0.0001) with a significant improvement in these indices following ELA-32 treatment (*P≤0.05, **** P≤0.0001, in comparison with MCT alone). ELA-32 alone had no effect on any parameter. ELA indicates Elabela/toddler; MCT, monocrotaline; LV, left ventricle; ns, not significantly different from saline control; PAH, pulmonary arterial hypertension; RV, right ventricle; and WGA, wheat germ agglutinin.