Abstract 13228: Nuclear AT1/AT2 Receptors Mediate Intracrine Angiotensin II Induced Fibrosis-related Gene Expression Changes in Atrial Fibroblasts
Background: Angiotensin II (Ang-II) is central to arrhythmia-promoting fibroblast (FB) remodeling. Ang-II action is classically attributed to cell membrane receptors/ second messengers, but there is increasing evidence of intracrine signaling via nuclear receptors. Here, we examined the nuclear localisation of AT1 (AT1R) and AT2 (AT2R) receptors and their potential functional role.
Methods: AT1R/AT2R localization was assessed in isolated canine atrial FBs by immunofluorescence and Western blot. Intact nuclei were purified by differential centrifugation. Nitric oxide (NO) production was measured via DAF-2 fluorescence, de novo RNA production by transcription initiation assay, and collagen1/3, MMP2 and fibrillin1 mRNA by qPCR. The expression of approximately 30,000 transcripts was analyzed by gene chip in isolated nuclei.
Results: AT1R and AT2R colocalized with Topro 3 and Lamin A/C in FB nuclei. Ang-II dose-dependently induced de novo RNA synthesis in isolated FB nuclei. AT1R (L-162,313) and AT2R (CGP 42112A) specific ligands enhanced transcription initiation in FB nuclei (219±15*; 148±8* cpm/ng DNA vs 69±9 control; n=5/group, *P<0.05). IP3R1 Ca2+-channels and eNOS colocalized with nuclear AT1R/AT2R. Ang-II enhanced FB nuclear NO production, which was reduced 85%* by the AT2R blocker PD123177 and 81%* by the NO synthase inhibitor L-NAME but unaffected by AT1R blocker losartan. AT2R-induced transcription initiation was reduced 79%* by L-NAME. AT1R-mediated transcription initiation was reduced 55%* by the IP3R-channel blocker 2-APB but not L-NAME. Ang-II exposure upregulated ECM-genes (collagen1 1.4-fold*, fibrillin1 2.0-fold*, laminin 1.8-fold*) in isolated atrial FB nuclei while others (MMP2 0.24-fold*; MMP9 0.59-fold*) were downregulated. Ang-II also upregulated important cell-signaling genes like PLC (3.8-fold*), IP3R1 (3.5-fold*), PDGF (2.5-fold*).
Conclusions: Our results indicate that intracellular Ang-II acts through nuclear receptors to regulate ECM and cell signaling genes fundamental to FB remodeling, coupling selectively to IP3R via AT1R and NO signalling via AT2R. These nuclear-delimited actions may be important in arrhythmogenic substrate development and could provide novel pharmacological targets.
- © 2013 by American Heart Association, Inc.