Qualitative and quantitative proteomic approach of epicardial cell secretome from atrial fibrillation patients after cholinergic stimulation
Authors: Couselo Seijas M , Molares Vila A , Bravo Lopez S B , Fernandez A L , Almenglo Buzon C , Fu X , Rodriguez Manero M , Eiras Penas S 
Published: October 2021

Abstract

Background
The association between epicardial adipose tissue (EAT) and atrial fibrillation (AF) was already described by several authors. In fact, botulinum toxin injection in this tissue reduces new-onset AF after open-heart surgery. Our previous results showed an elevation of adiposity in epicardial stromal vascular cells (SVC), assessed by the higher levels of fatty acid-binding protein 4 (FABP4) in AF patients; and a higher lipid accumulation in these cells after chronic acetylcholine treatment. We wanted to identify the proteins released from EAT SVC in presence/absence of mature adipocytes, that could be exerting a paracrine effect over the myocardium. The identification of these proteins might shed light on possible triggers in epicardial SVC and the mechanisms underlying botulinum toxin benefits on AF.

Material and methods
Proteomic studies were performed in 32 samples from 8 patients undergoing open-heart surgery (4 with and 4 without AF). Epicardial SVC were isolated by collagenase digestion and cultured in M199 medium. Then, cells were induced or not to adipocyte differentiation. After this intervention, cells were treated or not with acetylcholine (10uM) for 30 min. Conditioned medium was stored until be used. Differential released proteins were identified by nano-high performance liquid chromatography (HPLC) and Triple-Time of flying (TOF) analysis, and quantified by SWATH-Ms proteomics anaylisis.

Results
The quantitative proteomic approach has identified 111 common proteins in EAT SVC from patients with and without AF. A 79,3% of the genes which encoded the proteins identified were citoplasmic. A 78,4% were classified as components of the cellular exosomes, followed by genes related with centrosome (37,1%), nucleosome (15,5%), lysosomes (40,5%) and nucleolus (37,1%). Acute cholinergic treatment with ACh at 10 uM decreased α-defensin 3 (DEFA3, ID: 59666; p-value = 0,0297) secretion from EAT SVC of AF patients in comparison with EAT SVC from non-AF patients. In the same line, Peptidyl-prolyl cis-trans isomerase A (PPIA, ID: P62937) showed a lower secretion from SVC of AF patients (p=0,0326). After adipogenesis-induction, adipocyte presence modified the protein secretion under ACh treatment: Differences between AF and non-AF patients lied on 2 different proteins: profilin 1 (PFN1, ID: P07737, p-value = 0,0286) and β-enolase (ENO3, ID: P13929, p-value = 0,0414), showing a higher and lower secretion in AF patients regarding non-AF patients, respectively.

Conclusions
EAT SVC showed a differential protein secretion according adipocyte and AF presence. Although further studies are needed, the proteins differentially secreted in EAT SVC are related to inflammation (DEFA3), structure (PFN1) and glucose metabolism (ENO3), pointing the pathways that could be modified in AF patients.

Funding Acknowledgement
Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Plan Estatal de I+D+I 2016-2020 and ISCIII-Subdirecciόn General de Evaluaciόn y Fomento de la Investigaciόn el Fondo Europeo de Desarrollo Regional (FEDER) Figure 1. Common proteins secreted from EAT SVCFigure 2. Differentially secreted proteins in AF