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.

Figure 1. Role of Ang1-7 and Ang1-9 in AngII-induced hypertrophy

A, H9c2 cardiomyocytes were incubated with Ang1-7 or Ang1-9 at 10, 100 nm or 1 μm 30 min before stimulation with 100 nm AngII. Cells were then incubated for a further 96 h, fixed, stained with crystal violet and cell size measured using ImageProPlus. *P < 0.001 vs. control non-stimulated cells. #P < 0.05 vs. AngII-stimulated cells. B, RNA was isolated from H9c2 cardiomyocytes following 96 h exposure to 100 nm AngII and/or 100 nm Ang1-7 or Ang1-9, reverse transcribed and ANP, BNP, β-MHC and MLC expression quantified via real-time QPCR and normalised to 18S RNA expression. *P < 0.05 vs. control non-stimulated cells. C, following 96 h exposure to angiotensin peptides, α-actin filament reorganisation in H9c2 cardiomyocytes was evaluated by staining with FITC-labelled phalloidin. Magnification 100×, scale bar = 100 μm. D, left ventricular primary cardiomyocytes were extracted from adult rabbits via collagenase digestion, plated and immediately stimulated with Ang1-7 or Ang1-9 at 100 nm, 500 nm or 1 μm 30 min before stimulation with 500 nm AngII. Cells were then incubated for a further 24 h before measurement. Cell length and width were measured using ImageProPlus. *P < 0.01 vs. control non-stimulated cells. #P < 0.05 vs. AngII-stimulated cells.

Figure 2. Ang1-9 mediated inhibition of hypertrophy in the presence of ACE inhibition

A, H9c2 cardiomyocytes were incubated with captopril at 100 μm 15 min before addition of Ang1-9 (100 nm) and AngII. B, rabbit primary cardiomyocytes were incubated with captopril at 1 μm 15 min before addition of 500 nm Ang1-9. *P < 0.001 vs. control non-stimulated cells; #P < 0.001 vs. AngII stimulated cells.

Figure 3. The role of the Mas receptor in Ang1-7 and Ang1-9 signalling in cardiomyocytes

A, H9c2 cells were pre-incubated with AngII (100 nm), prior to addition of the Mas antagonist A779 (10 μm) 15 min before addition of 100 nm Ang1-7 or Ang1-9. B, addition of A779 (10 μm) in adult rabbit left ventricular primary cardiomyocytes, prior to addition of 500 nm Ang1-7 or Ang1-9. *P < 0.001 vs. control non-stimulated cells; #P < 0.001 compared to AngII stimulated cells.

Figure 4. The role of the AT₁R in Ang1-7 and Ang1-9 signalling in cardiomyocytes

H9c2 cardiomyocytes were stimulated with arginine vasopressin (1 μm) to induce hypertrophy. Losartan (1 and 10 μm) was added to block AngII-induced hypertrophy but not arginine vasopressin's action. Cells were pre-incubated with losartan and arginine vasopressin before addition of 100 nm Ang1-7 or Ang1-9. (VSP: arginine vasopressin). *P < 0.001 vs. control non-stimulated cells; #P < 0.001 vs. AngII stimulated cells.

Figure 5. The role of the AT₂R in Ang1-7 and Ang1-9 signalling in cardiomyocytes

A, AngII-stimulated H9c2 cardiomyocytes were pre-incubated with the AT₂R antagonist PD123,319 15 min before addition of Ang1-7 (100 nm) or Ang1-9 (100 nm). B, RNA was isolated from H9c2 cardiomyocytes following 96 h exposure to AngII (100 nm) and/or Ang1-7 (100 nm) or Ang1-9 (100 nm) in the presence or absence of the AT₂R antagonist PD123,319 (500 nm) before RNA was isolated, reverse transcribed and ANP and BNP expression quantified via real-time PCR. *P < 0.05 vs. control non-stimulated cells. C, in adult rabbit left ventricular primary cardiomyocytes PD123,319 was added 15 min prior to addition of Ang1-7 (500 nm) or Ang1-9 (500 nm). (PD123 = PD123,319.) *P < 0.001 vs. control non-stimulated cells; #P < 0.001 vs. AngII stimulated cells.

Figure 6. The role of the B₂R in Ang1-7 and Ang1-9 signalling in cardiomyocytes

H9c2 cardiomyocytes were incubated for 24 h in serum free medium before adding HOE140 (1 μm), A779 (10 μm) or PD123,319 (500 nm). Cells were incubated for 15 min before adding Ang1-7 or Ang1-9 (100 nm) followed by AngII 30 min later. Following 96 h incubation, cells were fixed and stained with crystal violet and cell size measured with ImageProPlus. *P < 0.001 vs. non-stimulated cells; #P < 0.001 vs. AngII stimulated cells; **P < 0.001 vs. Ang1-7 stimulated cells. (PD = PD123,319; HOE = HOE140.)