title: Angiotensin1-9 antagonises pro-hypertrophic signalling in cardiomyocytes via the angiotensin type 2 receptor.
abstract: The renin–angiotensin system (RAS) regulates blood pressure mainly via the actions of angiotensin (Ang)II, generated via angiotensin converting enzyme (ACE). The ACE homologue ACE2 metabolises AngII to Ang1-7, decreasing AngII and increasing Ang1-7, which counteracts AngII activity via the Mas receptor. However, ACE2 also converts AngI to Ang1-9, a poorly characterised peptide which can be further converted to Ang1-7 via ACE. Ang1-9 stimulates bradykinin release in endothelium and has antihypertrophic actions in the heart, attributed to its being a competitive inhibitor of ACE, leading to decreased AngII, rather than increased Ang1-7. To date no direct receptor-mediated effects of Ang1-9 have been described. To further understand the role of Ang1-9 in RAS function we assessed its action in cardiomyocyte hypertrophy in rat neonatal H9c2 and primary adult rabbit left ventricular cardiomyocytes, compared to Ang1-7. Cardiomyocyte hypertrophy was stimulated with AngII or vasopressin, significantly increasing cell size by approximately 1.2-fold (P < 0.05) as well as stimulating expression of the hypertrophy gene markers atrial natriuretic peptide, brain natriuretic peptide, β-myosin heavy chain and myosin light chain (2- to 5-fold, P < 0.05). Both Ang1-9 and Ang1-7 were able to block hypertrophy induced by either agonist (control, 186.4 μm; AngII, 232.8 μm; AngII+Ang1-7, 198.3 μm; AngII+Ang1-9, 195.9 μm; P < 0.05). The effects of Ang1-9 were not inhibited by captopril, supporting previous evidence that Ang1-9 acts independently of Ang1-7. Next, we investigated receptor signalling via angiotensin type 1 and type 2 receptors (AT1R, AT2R) and Mas. The AT1R antagonist losartan blocked AngII-induced, but not vasopressin-induced, hypertrophy. Losartan did not block the antihypertrophic effects of Ang1-9, or Ang1-7 on vasopressin-stimulated cardiomyocytes. The Mas antagonist A779 efficiently blocked the antihypertrophic effects of Ang1-7, without affecting Ang1-9. Furthermore, Ang1-7 activity was also inhibited in the presence of the bradykinin type 2 receptor antagonist HOE140, without affecting Ang1-9. Moreover, we observed that the AT2R antagonist PD123,319 abolished the antihypertrophic effects of Ang1-9, without affecting Ang1-7, suggesting Ang1-9 signals via the AT2R. Radioligand binding assays demonstrated that Ang1-9 was able to bind the AT2R (pKi = 6.28 ± 0.1). In summary, we ascribe a direct biological role for Ang1-9 acting via the AT2R. This has implications for RAS function and identifying new therapeutic targets in cardiovascular disease.
authors.affiliation: Institute of Cardiovascular and Medical Sciences, BHF GCRC, University of Glasgow, 126 University Place, Glasgow G12 8TA, UK.
authors.forename: M
authors.initials: M
authors.lastname: Flores-Muñoz
authors.forename: N J
authors.initials: NJ
authors.lastname: Smith
authors.forename: C
authors.initials: C
authors.lastname: Haggerty
authors.forename: G
authors.initials: G
authors.lastname: Milligan
authors.forename: S A
authors.initials: SA
authors.lastname: Nicklin
mesh_terms: D000803:Angiotensin I / Q000494:pharmacology / Q000502:physiology*; D000804:Angiotensin II / Q000494:pharmacology / Q000502:physiology*; D000818:Animals; D000831:Animals, Newborn; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D006367:HeLa Cells; D006801:Humans; D006984:Hypertrophy / Q000473:pathology; D032383:Myocytes, Cardiac / Q000473:pathology* / Q000502:physiology; D010446:Peptide Fragments / Q000494:pharmacology / Q000502:physiology*; D011817:Rabbits; D051381:Rats; D044139:Receptor, Angiotensin, Type 2 / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D010446:Peptide Fragments; D044139:Receptor, Angiotensin, Type 2; D000804:Angiotensin II; D000803:Angiotensin I