In vivo genetic profiling and cellular localization of apelin reveals a hypoxia-sensitive, endothelial-centered pathway activated in ischemic heart failure

Abstract

Signaling by the peptide ligand apelin and its cognate G protein-coupled receptor APJ has a potent inotropic effect on cardiac contractility and modulates systemic vascular resistance through nitric oxide-dependent signaling. In addition, there is evidence for counterregulation of the angiotensin and vasopressin pathways. Regulatory stimuli of the apelin-APJ pathway are of obvious importance but remain to be elucidated. To better understand the physiological response of apelin-APJ to disease states such as heart failure and to elucidate the mechanism by which such a response might occur, we have used the murine model of left anterior descending coronary artery ligation-induced ischemic cardiac failure. To identify the key cells responsible for modulation and production of apelin in vivo, we have created a novel apelin-lacZ reporter mouse. Data from these studies demonstrate that apelin and APJ are upregulated in the heart and skeletal muscle following myocardial injury and suggest that apelin expression remains restricted to the endothelium. In cardiac failure, endothelial apelin expression correlates with other hypoxia-responsive genes, and in healthy animals both apelin and APJ are markedly upregulated in various tissues following systemic hypoxic exposure. Experiments with cultured endothelial cells in vitro show apelin mRNA and protein levels to be increased by hypoxia, through a hypoxia-inducible factor-mediated pathway. These studies suggest that apelin-expressing endothelial cells respond to conditions associated with heart failure, possibly including local tissue hypoxia, and modulate apelin-APJ expression to regulate cardiovascular homeostasis. The apelin-APJ pathway may thus provide a mechanism for systemic endothelial monitoring of tissue perfusion and adaptive regulation of cardiovascular function.

Figure 1

Ischemic cardiac failure induces upregulation of apelin and APJ mRNA in the heart and skeletal muscle. Apelin (left column) and APJ (right column) mRNA levels (as measured by RT-PCR) in the heart (top row) increase as heart failure progresses, with significantly elevated levels by week 8 following ischemic injury (n=11 per group; bars represent means ± 95% CI; * P<0.001; **P<0.05). This pattern is consistent with known markers of heart failure in this model, such as β-natreutic peptide (Supplemental Figure 1). Apelin expression in the quadriceps muscle also increases, as early as week 4, reaching a peak by 8 weeks (* P<0.0001, **P<0.03). Quadriceps APJ is upregulated in a similar fashion following LAD ligation, but continues to increase through 12 weeks (*P<0.016, **P<0.0001, ***P<0.049). Pulmonary apelin and APJ levels (bottom row) are not significantly altered in ischemic heart failure.

Figure 2

Tissues from the transgenic apelin^+/lacZ reporter mouse demonstrate the apelin reporter gene is primarily expressed by the endothelium. Top two rows: staining for the apelin-LacZ reporter within the heart (left), quadriceps (middle), and lung (right column), reveals lacZ expression (blue) by vessels within the tissues. Endothelial cells of capillaries (black arrowheads) and veins (“V”) universally express the reporter, but arteries (“A”) do not. Bottom row: co-staining for LacZ reporter (blue) and the endothelial marker, CD31 (brown), confirms endothelial phenotype of apelin reporter-expressing cells (scale bars=25 µm). Three dimensional reconstruction (by serial confocal microscopy) of the same sections co-stained with fluorescent antibodies against LacZ and CD31 confirmed lacZ nuclei to be contained by endothelial cytosol (see Supplemental Videos 1 and 2).

Figure 3

Ischemic myocardial failure induced by LAD-ligation results in upregulation of the apelin reporter by coronary endothelium. A, Images show representative whole heart sections from transgenic apelin lacZ reporter mice 8 weeks following sham (left) and LAD ligation (right), scale bars=1 mm. Apelin reporter expression can be seen in surviving endothelium as well as neoangiogenic cells within the areas of myocardial infarct following LAD ligation (black-bordered inlays, scale bars=175µm). Intense Xgal staining is observed in endothelium remote from the infarct, suggesting increased apelin expression by capillary endothelial cells in the spared portions of the myocardium (red-bordered inlays, scale bars=25µm). B, LAD ligation leads to increased number of apelin reporter protein producing cells at sites remote from the infarction region. The graph depicts mean number of cells±SD over three general areas of interest: left ventricle (LV), septum (S), right ventricle (RV). Mean total cell counts from all areas (LV+S+RV) are given on the right. Cells were counted in a blinded fashion over 4–6 high power fields (20x) in each region of interest (n=2 hearts per group, *P<0.001).

Figure 4

LAD ligation-induced cardiac failure induces hypoxia of the skeletal muscle and upregulation of hypoxia-responsive genes. A, Oximetry measurements in quadriceps muscle demonstrate progressive hypoxia in murine peripheral skeletal muscle following LAD ligation compared to animals receiving sham procedure (n=5–7 animals per group, bars represent means±95%CI, *P<0.001). B, TNF-α, VEGF, and HIF-1α are significantly upregulated in the quadriceps muscle as heart failure progresses (n=11 per group; bars represent means ± 95% CI, P-values: *<0.045, **<0.0001, †<0.003, ††<0.009).

Figure 5

”Expression heat map” of selected parameters and gene expression changes measured in the 44 animal cohort undergoing LAD-ligation or sham procedure. The map represents correlations between the expression patterns of genes as well as cardiac function (FS). Notable positive correlations include concerted upregulation of hypoxia responsive genes (VEGF, HIF-1α, TNF-α) and apelin/APJ. Cardiac β-natriuretic peptide (BNP) is inversely correlated to cardiac function as is apelin. Color scale for degree of correlation is given beneath the table, with green representing a negative correlation and red positive. The lower half of the table presents the same information in numerical format, with significant correlations (r-values) depicted in blue (FS= fractional shortening; *P<0.05, **P<0.01).

Figure 6

The apelin-APJ pathway is upregulated in endothelial cells following systemic hypoxia in vivo. A, Apelin and APJ expression are significantly increased in the heart and lung following continual, systemic hypoxia (10% FiO2) for 1 week (n=11 per group; bars represent means ± 95% CI; *P<0.001). In quadriceps tissue, apelin mRNA levels showed a nearly two-fold mean increase, but this difference did not reaching statistical significance. B, Representative histological sections of heart, lung, and quadriceps from apelin^+/lacZ reporter mice kept under ambient normoxia (21% FiO₂, top row) or hypoxia (10% FiO₂) for 1 week, demonstrating that apelin reporter expression remains restricted to the endothelium and suggesting that increased tissue mRNA levels correlate to increased apelin expression by endothelial cells (scale bars=20 µm).

Figure 7

Hypoxia-induced apelin upregulation in human cultured human endothelial cells. A, Twenty-four hour hypoxic exposure (1%FiO₂) resulted in significant upregulation of apelin by coronary artery endothelial cells (HCAEC), dermal microvascular endothelial cells (HMVEC-D), pulmonary artery endothelial cells (HPAEC) and ECV-304 cells in vitro (*P<0.005). B, Soluble apelin production (as determined by ELISA) by HCAEC is significantly increased following hypoxic exposure. C, Upregulation of apelin mRNA in cultured HEK293 cells transfected with constitutively active HIF-1α (HIF-ODD) and HIF-2α (*P<0.04, **P<0.003). In all panels, bars represent mean±SD from 2–5 representative experiments.