Asn111, localized in the third transmembrane domain of the AT1 receptor for angiotensin II, plays a critical role in stabilizing the inactive conformation of the receptor. We evaluated the functional and G protein-coupling properties of mutant AT1 receptors in which Asn111 was substituted with smaller (Ala or Gly) or larger residues (Gln or Trp). All four mutants were expressed at high levels in COS-7 cells and, except for N111W-AT1, recognized 125I-Ang II with high affinities comparable to that of the wild-type AT1 receptor. In phospholipase C assays, the four mutants encompassed the entire spectrum of functional states, ranging from constitutive activity (without agonist) for N111A-AT1 and N111G-AT1 to a significant loss of activity (upon maximal stimulation) for N111Q-AT1 and a major loss of activity for N111W-AT1. In Ca2+ mobilization studies, N111W-AT1 produced a weak Ca2+ transient and, unexpectedly, N111G-AT1 also produced a Ca2+ transient that was much weaker than that of the wild-type AT1. The agonist binding affinity of N111W-AT1 was not modified in the presence of GTPγ S, suggesting that this receptor is not basally coupled to a G protein. GTPγ S did not modify the high agonist-binding affinity of N111G-AT1 but abolished the coimmunoprecipitation of Gq/11α with this constitutively active mutant receptor. These results are a direct demonstration that the N111G-AT1 receptor maintains a high affinity conformation despite being uncoupled from the G protein Gq/11.

Objectives: Arterial hypertension, when exacerbated by excessive dietary salt intake, worsens the morbidity and mortality rates associated with cardiovascular and renal diseases. Stimulation of the apelinergic system appears to protect against several circulatory system diseases, but it remains unknown if such beneficial effects are conserved in severe hypertension. Therefore, we aimed at determining whether continuous infusion of apelinergic ligands (i.e., Apelin-13 and Elabela) exerted cardiorenal protective effects in spontaneously hypertensive (SHR) rats receiving high-salt diet.Methods: A combination of echocardiography, binding assay, histology, and biochemical approaches were used to investigate the cardiovascular and renal effects of Apelin-13 or Elabela infusion over 6 weeks in SHR fed with normal-salt or high-salt chow.Results: High-salt intake upregulated the cardiac and renal expression of APJ receptor in SHR. Importantly, Elabela was more effective than Apelin-13 in reducing high blood pressure, cardiovascular and renal dysfunctions, fibrosis and hypertrophy in high-salt fed SHR. Unlike Apelin-13, the beneficial effects of Elabela were associated with a counter-regulatory role of the ACE/ACE2/neprilysin axis of the renin-angiotensin-aldosterone system (RAAS) in heart and kidneys of salt-loaded SHR. Interestingly, Elabela also displayed higher affinity for APJ in the presence of high salt concentration and better resistance to RAAS enzymes known to cleave Apelin-13.Conclusion: These findings highlight the protective action of the apelinergic system against salt-induced severe hypertension and cardiorenal failure. As compared with Apelin-13, Elabela displays superior pharmacodynamic and pharmacokinetic properties that warrant further investigation of its therapeutic use in cardiovascular and kidney diseases.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2–related MD, and collagen VI–related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13–amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.