Intracellular Angiotensin‐II Interacts With Nuclear Angiotensin Receptors in Cardiac Fibroblasts and Regulates RNA Synthesis, Cell Proliferation, and Collagen Secretion

Abstract

Background Cardiac fibroblasts play important functional and pathophysiological roles. Intracellular (“intracrine”) angiotensin‐ II (Ang‐ II ) signaling regulates intercellular communication, excitability, and gene expression in cardiomyocytes; however, the existence and role of intracrine Ang‐ II signaling in cardiac fibroblasts is unstudied. Here, we evaluated the localization of Ang‐ II receptors on atrial fibroblast nuclei and associated intracrine effects of potential functional significance. Methods and Results Immunoblots of subcellular protein‐fractions from isolated canine atrial fibroblasts indicated the presence of nuclear Ang‐II type 1 receptors ( AT 1Rs) and Ang‐II type 2 receptors ( AT 2Rs). Fluorescein isothiocyanate–Ang‐ II binding displaceable by AT 1R‐ and AT 2R‐blockers was present on isolated fibroblast nuclei. G‐protein subunits, including Gαq/11, Gαi/3, and Gβ, were observed in purified fibroblast nuclear fractions by immunoblotting and intact‐fibroblast nuclei by confocal immunocytofluorescence microscopy. Nuclear AT 1Rs and AT 2Rs regulated de novo RNA synthesis ([α 32 P] UTP incorporation) via IP 3R‐ and NO ‐dependent pathways, respectively. In intact cultured fibroblasts, intracellular Ang‐ II release by photolysis of a membrane‐permeable caged Ang‐ II analog led to IP 3R‐dependent nucleoplasmic Ca 2+ ‐liberation, with IP 3R3 being the predominant nuclear isoform. Intracellular Ang‐ II regulated fibroblast proliferation ([ 3 H]thymidine incorporation), collagen‐1A1 mRNA ‐expression, and collagen secretion. Intracellular Ang‐ II and nuclear AT 1R protein levels were significantly increased in a heart failure model in which atrial fibrosis underlies atrial fibrillation. Conclusions Fibroblast nuclei possess AT 1R and AT 2R binding sites that are coupled to intranuclear Ca 2+ ‐mobilization and NO liberation, respectively. Intracellular Ang‐ II signaling regulates fibroblast proliferation, collagen gene expression, and collagen secretion. Heart failure upregulates Ang‐ II intracrine signaling‐components in atrial fibroblasts. These results show for the first time that nuclear angiotensin‐ II receptor activation and intracrine Ang‐ II signaling control fibroblast function and may have pathophysiological significance.