APJ acts as a dual receptor in cardiac hypertrophy

Abstract

Cardiac hypertrophy is initiated as an adaptive response to sustained overload but progresses pathologically as heart failure ensues. Here we report that genetic loss of APJ, a G-protein-coupled receptor, confers resistance to chronic pressure overload by markedly reducing myocardial hypertrophy and heart failure. In contrast, mice lacking apelin (the endogenous APJ ligand) remain sensitive, suggesting an apelin-independent function of APJ. Freshly isolated APJ-null cardiomyocytes exhibit an attenuated response to stretch, indicating that APJ is a mechanosensor. Activation of APJ by stretch increases cardiomyocyte cell size and induces molecular markers of hypertrophy. Whereas apelin stimulates APJ to activate Gαi and elicits a protective response, stretch signals in an APJ-dependent, G-protein-independent fashion to induce hypertrophy. Stretch-mediated hypertrophy is prevented by knockdown of β-arrestins or by pharmacological doses of apelin acting through Gαi. Taken together, our data indicate that APJ is a bifunctional receptor for both mechanical stretch and the endogenous peptide apelin. By sensing the balance between these stimuli, APJ occupies a pivotal point linking sustained overload to cardiomyocyte hypertrophy.

Figure 1. APJ-KO mice are protected from hypertrophy after TAC

a, Anatomical view and b, Histological sections of WT and APJ-KO mice 90 days after surgery. c, Cell membrane staining (wheat germ agglutinin). d, Quantification from (c). e, Trichrome staining (fibrosis in blue, stars). f, quantification of (e). g, Fractional shortening (%FS) decreased in WT mice after TAC, but did not change significantly in the APJ-KO mice. APJ-KO mice fail to develop heart failure upon sustained TAC as shown by echocardiographyc analysis. h, Heart weight-to-body weight ratio (HW/BW) at baseline and in TAC operated mice, 90 days after surgery (see Suppl. Table 4 for details). Error bars are SEM.*p<0.05 between indicated groups, ANOVA.

Figure 2. APJ mediates a stretch response that can be modulated by apelin

a–b, Representative force measurements (arbitrary units) for end-diastolic and end-systolic length-tension relationships in adult cardiomyocytes from WT (a) and APJ-KO (b) mice plotted against diastolic length (normalized to unstretched length). Cells were paced at 1Hz. c, Frank-Starling Gain (FSG) attained by dividing the active force by the passive force from experiments in (a) and (b) plotted as a function of diastolic length (n= 8 WT and 7 APJ-KO cardiomyocytes). d, Average FSG (at 1.02 sarcomeric length from c) is shown for APJ-KO and WT both with (+) and without (−) 10 nM apelin administration (n=6 for WT+apelin and n=7 for APJ-KO+apelin). Error bars are SEM.

Figure 3. Stretch activation of APJ enhances β-arrestin while reducing G-protein signaling

a, Immunoblot and b, Quantification of ERK from APJ stably transfected (APJ-HEK) and parental (HEK) cells treated for 5 minutes with 100 nM apelin or stretch in absence or presence of PTX (Gα_i inhibitor), n=3. c, Effect of 1 μM apelin or d, stretch on cAMP levels. 1 μM isoproterenol (iso) was used to artificially elevate cAMP to study Gα_i activation, n= 4 (PTX). e, G-protein (Gα_q,s,i and_12/13) activation by apelin and effect of stretch (red) in CHO cells expressing APJ and Gα₁₆. Receptor stimulation activated the promiscuous Gα₁₆, phospholipase-Cβ and caused the accumulation of IP1 (representative of 3 experiments, n=4 samples). f–g, Arrestin recruitment to the APJ receptor in response to apelin (black) and stretch (red) by an enzyme complementation assay in CHO cells expressing recombinant APJ and β-arrestin2 (representative of 3 experiments, n=3 samples). (f) Represents full range of β-arrestin binding to APJ under either physiological or pharmacological doses of apelin. (g) Shows the data points for 0 – 10⁻¹⁰ M apelin. RLU= Relative light units. Error bars represent SEM. *p<0.05 between indicated groups, ANOVA.

Figure 4. APJ activation through mechanical stretch elicits cardiac hypertrophy

a–l, ANF immunostaining (white) and nuclear DAPI staining (blue) of rat neonatal ventricular cardiomyocytes transduced with rat APJ (Ad-APJ-GFP) or control GFP (Ad-GFP, green). m, Quantification of from a-l (n=250–350 cells). n, qPCR analysis of the ratio between β– and α–MHCs, as an independent index of hypertrophy, n=3–5 samples. o, Mean cell sizes in a-l, n=24 cells. p, Responsiveness of transfected cells to apelin treatment, n=4–5. q, ANF expression in cardiomyocytes in the presence of conditioned-media from stretched cardiomyocytes (str. media), n=3. r, Apelin ELISA of conditioned media (12 h) from cardiomyocytes (CM) non-stretched and stretched. s, Apelin standard curve of CHO APJ-β-arrestin interaction assay; red arrows represent the response elicited by 2 samples from (r) t–v. Higher magnification image of NRVC showing ANF (white) and APJ (green) expression. White arrow indicates APJ⁻ cells not expressing ANF. w, Gα_i inhibition with PTX blocked the ability of apelin to prevent ANF expression, n=5. x-y, qRT-PCR for ANF in cells treated with (x) or without (y) inhibitors, n=3. z, Diminished expression of hypertrophy markers in Ad-APJ-GFP cardiomyocytes transfected upon knockdown of βarrestin1 (siβARR1), βarrestin2 (siβARR2) or both (siβARR1+2), n=4. Except for (p) and (y) all experiments were performed in the presence of inhibitors of AT-1 (candesartan, 100 nM) and ET-A (BQ123, 300 nM) added one hour prior to stretch and/or apelin treatment until fixation. All are representative experiments performed at least three independent times. Error bars are SEM. *p<0.05 between indicated groups, ANOVA.