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.

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.

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.

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.