Modulation of the apelin/APJ system in heart failure and atherosclerosis in man

Abstract

Background and purpose: The aim of this study was to determine whether the apelin/APJ system is altered in human cardiovascular disease by investigating whether the expression of apelin or its receptor is altered at the protein level.

Experimental approach: Radioligand binding studies were used to determine apelin receptor density in human cardiac tissues. Apelin peptide levels in cardiovascular tissues were determined by radioimmunoassay. In vitro pharmacology was used to assess vasoactive properties of apelin in human coronary artery. Localization of apelin and its receptor in coronary artery was determined using immunohistochemistry.

Key results: Apelin receptor density was significantly decreased in left ventricle from patients with dilated cardiomyopathy or ischaemic heart disease compared with controls, but apelin peptide levels remained unchanged. Apelin was up-regulated in human atherosclerotic coronary artery and this additional peptide localized to the plaque, colocalizing with markers for macrophages and smooth muscle cells. Apelin potently constricted human coronary artery.

Conclusions and implications: We have detected changes in the apelin/APJ system in human diseased cardiac and vascular tissue. The decrease in receptor density in heart failure may limit the positive inotropic actions of apelin, contributing to contractile dysfunction. The contribution of the increased apelin levels in atherosclerotic coronary artery to disease progression remains to be determined. These data suggest a potential role for the apelin/APJ system in human cardiovascular disease.

Figure 1

[Glp⁶⁵,Nle⁷⁵,Tyr⁷⁷][¹²⁵I]-apelin-13 binding in human left ventricle. (A) Specificity of [Glp⁶⁵,Nle⁷⁵,Tyr⁷⁷][¹²⁵I]-apelin-13 binding (mean ± SEM, n = 3). ANP, atrial natriuretic peptide. (B) Time-dependent association of [Glp⁶⁵,Nle⁷⁵,Tyr⁷⁷][¹²⁵I]-apelin-13 binding; a representative curve is shown, with an association rate constant (K_obs) of 0.038 min⁻¹. (C) Time-dependent dissociation of [Glp⁶⁵,Nle⁷⁵,Tyr⁷⁷][¹²⁵I]-apelin-13 binding; a representative curve is shown, with a dissociation rate constant (K₋₁) of 0.023 min⁻¹. (D) Example saturation binding curve with a dissociation constant (K_D) of 0.29 nM, maximal receptor density (B_max) of 0.55 fmol·mg⁻¹ protein and a Hill coefficient (n_H) of 1.03. (E) Maximal receptor density (B_max) in left ventricle from control hearts (n = 7) and patients with dilated cardiomyopathy (DCM, n = 6) or ischaemic heart disease (IHD, n = 8); horizontal lines represent median values; *P < 0.05; #P < 0.01. DCM, dilated cardiomyopathy; IHD, ischaemic heart disease.

Figure 2

(A) Typical standards curve (solid symbols) and dilutions of an atrial tissue sample (open symbols); expressed as mean of duplicate determinations. (B) Apelin-like immunoreactivity (LI) levels in human cardiac tissue and coronary artery (n = 5–6). CA, coronary artery; DCM, dilated cardiomyopathy; IHD, ischaemic heart disease; LV, left ventricle. *P < 0.01.

Figure 3

(A) Western blot using rabbit anti-APJ(357–377) showing a single band of approximately 60 kDa in human atherosclerotic coronary artery (CA, n = 3), attenuated following pre-absorption with the immunizing peptide. (B–H) Representative photomicrographs showing APJ-like immunoreactivity (LI) in human histologically normal (B, C) and atherosclerotic (D–H) coronary artery. (C, E) Primary antibody omitted. APJ-LI within the atherosclerotic plaque colocalizes with smooth muscle α-actin (SMαA)-LI (F) and CD68-LI (G), but not with CD3-LI (H). All n = 3. Scale bar 200 µm (B–E) or 50 µm (F–H).

Figure 4

Representative photomicrographs showing apelin-like immunoreactivity (LI) in human histologically normal (A, B) and atherosclerotic (C–G) coronary artery. (B, D) Primary antibody omitted. Within the atherosclerotic plaque apelin-LI colocalizes with smooth muscle α-actin (SMαA)-LI (E) and CD68-LI (F), but not with CD3-LI (G). All n = 3. Scale bar 200 µm (A–D) or 50 µm (E–G). Arrow indicates endothelial staining.

Figure 5

Representative cumulative concentration-response curve to (Pyr¹)apelin-13 in human isolated coronary artery. Arrows indicate time points at which doses were added.