Endogenous regulation of cardiovascular function by apelin-APJ

Abstract

Studies have shown significant cardiovascular effects of exogenous apelin administration, including the potent activation of cardiac contraction. However, the role of the endogenous apelin-APJ pathway is less clear. To study the loss of endogenous apelin-APJ signaling, we generated mice lacking either the ligand (apelin) or the receptor (APJ). Apelin-deficient mice were viable, fertile, and showed normal development. In contrast, APJ-deficient mice were not born in the expected Mendelian ratio, and many showed cardiovascular developmental defects. Under basal conditions, both apelin and APJ null mice that survived to adulthood manifested modest decrements in contractile function. However, with exercise stress both mutant lines demonstrated consistent and striking decreases in exercise capacity. To explain these findings, we explored the role of autocrine signaling in vitro using field stimulation of isolated left ventricular cardiomyocytes lacking either apelin or APJ. Both groups manifested less sarcomeric shortening and impaired velocity of contraction and relaxation with no difference in calcium transient. Taken together, these results demonstrate that endogenous apelin-APJ signaling plays a modest role in maintaining basal cardiac function in adult mice with a more substantive role during conditions of stress. In addition, an autocrine pathway seems to exist in myocardial cells, the ablation of which reduces cellular contraction without change in calcium transient. Finally, differences in the developmental phenotype between apelin and APJ null mice suggest the possibility of undiscovered APJ ligands or ligand-independent effects of APJ.

Fig. 1.

Targeting of the apelin and APJ loci. A: a construct encoding a nuclear targeted lacZ expression cassette was integrated by homologous recombination into the apelin locus, resulting in deletion of the first exon including the native ATG and leader peptide. Transfections were performed in 129SVJ embryonic stem cells, and targeted cells were microinjected into C57Bl/6 blastocysts. B: germline transmission of the apelin targeted allele in male mice. Male agouti offspring of chimeric founder mice were evaluated by Southern blot with an apelin genomic probe. The wild-type allele (+) migrates at 12 kb, and the targeted allele (−) migrates at 17 kb. There is only one allele for apelin in these male mice since apelin is on the X chromosome. C: a construct was created with loxP recombination sites flanking the APJ gene, and a loxP flanked neomycin resistance (neo) gene 3′ to the APJ gene. D: germline transmission of the APJ targeted allele was evaluated by PCR. The wild-type band (+) is 100 bp, and the targeted band (−) is 225 bp.

Fig. 2.

Imaging studies reveal modest differences in load-dependent measures of contractility. A: APJ null mice have significantly decreased fractional shortening by echocardiography (APJ^+/+, 44.9 ± 2.7% and APJ^−/−, 31.3 ± 1.6%; P = 0.0002, n = 12/group). B: apelin null mice reveal a nonsignificant trend toward decreased contractility using fractional shortening by echocardiography (apelin^+/+, 40.4 ± 1.1% and apelin^−/−, 37.9 ± 0.8%; P = 0.09, n = 13/group) and ejection fraction (C) by magnetic resonance imaging (apelin^+/+, 87.9 ± 1.5%, n = 8 and apelin^−/−, 83.9 ± 2.9%; P = 0.2, n = 7/group). *P < 0.05.

Fig. 3.

Apelin null mice have decreased intrinsic contractility and APJ null mice trend toward decreased contractility as measured by load-independent parameters. There was a significantly lower end-systolic pressure volume relationship (ESPVR) in the apelin null mice [A; apelin^+/+, 11.1 ± 0.73 and apelin^−/−, 6.9 ± 0.56 mmHg/relative volume units (RVU); P = 0.0002] and a trend toward decreased ESPVR in the APJ null mice (B; APJ^+/+, 10.2 ± 0.59 and APJ^−/−, 8.9 ± 0.51 mmHg/RVU; P = 0.15). Similar trends were seen with preload recruitable stroke work (PRSW) in the apelin null mice (C; apelin^+/+, 44.1 ± 3.2 and apelin^−/−, 36.5 ± 3.7 mmHg × RVU; P = 0.13) and APJ null mice (D; APJ^+/+, 39.0 ± 3.7 and APJ^−/−, 32.0 ± 3.4 mmHg × RVU; P = 0.19) as well as with maximal elastance (E_max) in the apelin null mice (E; apelin^+/+, 25.2 ± 1.9 and apelin^−/−, 20.9 ± 1.7 mmHg/RVU; P = 0.11) and APJ null mice (F; APJ^+/+, 25.6 ± 1.6 and APJ^−/−, 22.9 ± 2.7 mmHg × RVU; P = 0.38). Representative pressure-volume loops of apelin null (G) and wild-type (H) mice are shown. *P < 0.05.

Fig. 4.

Apelin and APJ null mice have reduced exercise capacity and apelin null mice have lower maximal oxygen consumption (V̇o₂). Graded treadmill running reveals that apelin (A) and APJ (B) null mice have impaired exercise capacity (apelin^+/+, 25.65 ± 0.77 and apelin^−/−, 23.31 ± 0.59 min, P = 0.034; and APJ^+/+, 23.64 ± 0.74 and APJ^−/−, 20.30 ± 0.97 min, P = 0.009). C: apelin null mice have lower maximal oxygen consumption (apelin^+/+, 6269.2 ± 116 and apelin^−/−, 5885.0 ± 120 ml·kg⁻¹·h⁻¹; P = 0.033). D: APJ null mice have similar maximal oxygen consumption [APJ^+/+, 6,534 ± 162 and APJ^−/−, 6,740 ± 329 ml·kg⁻¹·h⁻¹; P = not significant (ns)]. E: apelin null mice have a trend toward lower resting oxygen consumption (apelin^+/+, 4,240 ± 124 and apelin^−/−, 3,944 ± 107 ml·kg⁻¹·h⁻¹; P = 0.09). F: mean oxygen consumption over time in apelin null (n = 12) and wild-type (n = 17) mice during treadmill exercise. *P < 0.05.

Fig. 5.

Apelin and APJ null cardiomyocytes show decreased contractile function. Sarcomeric shortening was significantly decreased in the apelin null myocytes (A; apelin^+/+, 4.52 ± 0.26% and apelin^−/−, 3.69 ± 0.23%; P = 0.02) and APJ null myocytes (B; APJ^+/+, 6.98 ± 0.40% and APJ^−/−, 5.07 ± 0.41%; P = 0.002). The speed of contraction was also significantly reduced in the apelin null myocytes (C; apelin^+/+, −1.10 ± 0.06 and apelin^−/−, −0.89 ± 0.05 μm/s; P = 0.011) and the APJ null myocytes (D; APJ^+/+, −2.41 ± 0.15 and APJ^−/−, −1.77 ± 0.14 μm/s; P = 0.003). The velocity of relaxation was lower in the apelin null myocytes (E; apelin^+/+, 0.65 ± 0.06 and apelin^−/−, 0.51 ± 0.04 μm/s; P = 0.04) and the APJ null myocytes (F; APJ^+/+, 1.63 ± 0.12 and APJ^−/−, 1.07 ± 0.11 μm/s; P = 0.001). G: representative raw loop of sarcomeric function demonstrating decreased contractility of cardiomyocytes from the apelin null mice. *P < 0.05.

Fig. 6.

Apelin and APJ null cardiomyocytes show similar intracellular calcium kinetics and troponin I phosphorylation. There were similar calcium transients in the apelin null myocytes (A; apelin^+/+, 31.0 ± 2.6% and apelin^−/−, 34.0 ± 2.5%; P = NS) and APJ null myocytes (B; APJ^+/+, 38.1 ± 2.4% and APJ^−/−, 37.6 ± 2.6%; P = NS). C: phosphorylation levels of troponin I at serine residues 23–24 were similar at basal conditions between the apelin null and wild-type mice as expressed by the ratio of phos-troponin I (phos-trop I) to troponin I (trop I; apelin^+/+, 0.69 ± 0.03 and apelin^−/−, 0.64 ± 0.03; P = NS).