β1-adrenergic receptor autoantibodies from heart failure patients enhanced TNF-α secretion in RAW264.7 macrophages in a largely PKA-dependent fashion

Autoantibodies against the second extracellular loop of beta 1-adrenergic receptor (beta 1-AA) not only contribute to increased susceptibility to heart failure, but also play a causative role in myocardial remodeling through their catecholamine-like effects via binding with the beta 1-adrenergic receptor. The current study was designed to determine whether beta 1-AA isolated from the sera of heart failure patients could cause TNF-a secretion from the murine macrophage-like cell line RAW264.7. Blood samples were collected from 40 patients who had suffered heart failure, as well as from 40 healthy subjects. The titer of beta 1-AA and the level of TNF-a were detected using ELISA. The effect of beta 1-AA on murine macrophage-like cell line RAW264.7 proliferation was detected by CCK-8 kits and CFSE assay. Western blot assay was used to analyze the expression of phospho-VASP. beta 1-AA appeared more frequently in patients with heart failure than in healthy subjects. The beta 1-AA isolated from heart failure patients promoted an increase of TNF-a levels, which could be completely blocked by the selective beta 1-adrenergic receptor antagonist metoprolol and the second extracellular loop of beta 1-adrenergic receptor (beta 1-AR-ECII), but only partially inhibited by PKA inhibitor H89. Furthermore, the beta 1-AA could enhance the proliferation of RAW264.7 cells in vitro. Meanwhile, the expression of phospho-VASP was markedly increased in the presence of beta 1-AA. These results demonstrate for the first time that the beta 1-AA isolated from heart failure patients could bind with beta 1-AR on the surface of RAW264.7 cells, causing the release of TNF-a largely in a PKA-dependent fashion. J. Cell. Biochem. 113: 32183228, 2012. (c) 2012 Wiley Periodicals, Inc.