FPR2 agonists exert cardioprotection by resolving inflammatory signalling in macrophages, not cardiomyocytes or cardiac fibroblasts

Background Persistent inflammation drives the pathogenesis and progression of atrial fibrillation (AF) through activation of the NLRP3 inflammasome 1. Lipoxin A4 (LxA4), Resolvin D1 (RvD1), and BMS-986235 (BMS) (a synthetic agonist) are specialized pro-resolving molecules that signal via formyl peptide receptor 2 (FPR2) to resolve inflammation. These molecules are protective in in vivo models of heart disease 2-4. Pathological inflammatory signalling occurs in cardiomyocytes (CMs) and cardiac fibroblasts (FBs), along with immune cells. However, the cell type-specific signalling underlying FPR2 agonist cardioprotective function is unknown. Purpose To define cell-type specific signalling mediating FPR2 agonist cardioprotective actions. Methods Atrial CMs and FBs were isolated from adult human, dog (24h atrial tachypaced (AT) CMs), and rat (MYL4) AF models. Blood monocytes were isolated and differentiated into macrophages in vitro. Cells were treated with FPR2 agonists, lipopolysaccharide (LPS), and angiotensin II (Ang II). FPR2 and NLRP3 inflammasome components were assessed by digital and quantitative PCR, Western immunoblotting, and confocal microscopy; extracellular IL-1β release by ELISA. Fibroblast activation was quantified by smooth muscle actin (SMA) fibre formation and Ki67 nuclear expression. Results FPR2 transcript and protein levels were significantly increased* (*P < 0.05) in human and rat AF CMs and FBs compared to sinus rhythm (SR) (Fig 1A, B). Only FPR2 protein levels were decreased* in dog AT CMs (Fig 1C). NLRP3, IL-1β, and PYCARD expression was increased* in AF CMs (Fig 1D). NLRP3 and IL-1β were elevated* in rat AF FBs (Fig 1E). Dog AT CMs secreted increased levels* of extracellular IL-1β (Fig 1F). Only LxA4 reduced* dog AT CM transcript levels of PYCARD and extracellular IL-1β release (Fig 2A). FPR2 agonists did not affect CM or FB inflammatory signalling in humans and rats. FPR2 agonists did not attenuate LPS-induced IL-6 and NLRP3 gene expression or extracellular IL-1β release (Fig 2C) from CMs. FB SMA fibre formation and Ki67 nuclear expression induced by Ang II treatment was not reduced by FPR2 agonists (Fig 2D). However, RvD1 and BMS reduced* inflammatory signalling in macrophages by attenuating IL-1β gene expression and IL-1β release (Fig 2E, F). Conclusions FPR2 agonists do not work directly on CMs and FBs to attenuate inflammation in AF. Rather, they exert direct protective effects via innate immune cells. Our findings suggest that macrophages mediate the cardioprotective inflammation resolution effects of FPR2 agonists.