Abstract 10257: Nuclear Angiotensin-II Receptor Gene Expression in Cardiac Fibroblasts: Receptor Subtype-Specific Differential Role of IP-3-Receptor (IP3R) Related and Nitric Oxide Signaling
Introduction: Angiotensin-II (Ang-II) is a crucial regulator of cardiac fibroblast (FB) structure and function. This study assessed whether activation of nuclear delimited AT1 (AT1R) and AT2 (AT2R) receptors in cardiac fibroblasts activates signalling events that influence gene expression.
Methods: Canine atrial (Atr) and ventricular (Vt) FBs were isolated and intracellular localization of AT1R and AT2R assessed by immunofluorescence and Western blot. Intact nuclei were purified by differential centrifugation. Nitric oxide (NO) was detected with the fluorescent dye DAF-2. De novo RNA production was measured by transcription initiation assay ([α32P]UTP).
Results: AT1R and AT2R proteins localized to nuclear membrane (immunohisto and immunoblot) and colocalized with nuclear markers TOPRO 3 and Histone 3. Purified nuclei contained high concentrations of DNA (Atr nuclei 2206±108* μ g/ml DNA vs cytosol 97±13; Vt nuclei 874±20* μ g/ml DNA vs cytosol 84±8, *P<0.001) and were stained by nucleic acid marker Draq5, but not by the endosomal marker AlexaFluor 594-transferrin. FB nuclei loaded with DAF-2 showed increased NO production with Ang-II, which was reduced by 85%* (Atr) and 77%* (Vt) by the AT2R blocker PD123177 and 81%* (Atr) and 74%* (Vt) by the NO synthase blocker L-NAME, but not by AT1R blocker losartan. AT1R (L-162,313) and AT2R (CGP 42112A) ligands enhanced transcription initiation in Atr and Vt FB nuclei (Atr: 219±15+ cpm/ng DNA and 148±8+ cpm/ng DNA vs 69±9 cpm/ng DNA control; Vt: 260±21+ cpm/ng DNA and 240±6+ cpm/ng DNA vs 49±5 cpm/ng DNA control, n=5/group, +P<0.05). AT1R-mediated transcription initiation responses were reduced by 55%+ (Atr) and 48%+ (Vt) by the IP3R-blocker 2APB (but not L-NAME) and AT2R-mediated responses were reduced by 79%+ (Atr) and 29%+ (Vt) by L-NAME. Atr nuclei pretreated with both 2APB and L-NAME prior to Ang-II had 91%* reductions in [α32P]UTP incorporation, similar to pretreatment with the RNA polymerase II inhibitor α-amanitin.
Conclusions: Ang-II regulates gene expression in cardiac FBs through activation of nuclear AT1 and AT2 receptors, with IP3R activation and NO generation being respective effectors. FB nuclear ATR-signaling may regulate FB phenotype and play an important role in cardiac fibrosis and remodeling.
- © 2011 by American Heart Association, Inc.