Study population: the study population included 105 patients, without history of paroxysmal or permanent atrial fibrillation, undergoing elective surgery for CABG or valve replacement for severe aortic stenosis at the cardiac surgery unit of University of Naples “Federico II”. The presence of chronic inflammatory diseases and/or cancer represented exclusion criteria, given their association with systemic and/or visceral fat inflammation. Demographic and clinical data including drug therapies were collected from all patients. The study protocol was compliant to the ethical guidelines of the 1975 Declaration of Helsinki. All the study procedures received approval by our institution's human research committee (Protocol n.301/19). All patients provided written informed consent before their inclusion into the study.
Transthoracic Echocardiography: before cardiac surgery, all patients underwent complete echocardiographic study, performed with a VIVID E9 (GE Healthcare) machine. In addition to the standard parameters, the maximum EAT thickness was evaluated, from the parasternal long-axis view, at end systole, between the right ventricle and the ascending aorta (13). Measurements of EAT thickness were performed offline by two independent blinded echocardiographers. The average value from three cardiac cycles was used for the statistical analysis.
Tissues and serum collection: serum samples and EAT biopsies were collected from all patients undergoing cardiac surgery before the cardiopulmonary bypass (CPB). EAT biopsy samples (average 0.1 to 0.5 g) were taken between the free wall of the right ventricle and the anterior surface of the ascending aorta. EAT secretomes were obtained as follows: tissues were weighted, cut into small pieces, and transferred into a 12-well plate. According to tissue weight, serum-free Dulbecco modified Eagle medium (DMEM) (1 mL medium/0.1 g tissue) was added to the well and incubated at 37°C in a CO2 incubator. After 24 h, medium was collected and centrifuged at 14,000g to remove debris and analyzed for cytokines content, as described below.
Serum and EAT conditioned media were screened for the concentration of IL-1β, IL-1ra, IL-6, IL-8, IL-13, basic Fibroblast Growth Factor (FGF), Interferon (IFN)-γ, Monocyte Chemoattractant Protein (MCP)-1, Regulated on Activation Normal T-cell Expressed and Secreted (RANTES/CCL5), and Tumor Necrosis Factor (TNF)-α, using the Bio-Plex multiplex Human Cytokine and Growth factor kits (Bio-Rad) according to the manufacturer's protocol.
Materials: Media were from Lonza (Lonza Group Ltd., Basel, Switzerland).
ECG monitoring: after surgery, heart rate and rhythm were monitored for 7 days, by continuous telemetry (ApexPro 7-lead system; General Electric Medical Systems), at the cardiac intensive care unit. Atrial fibrillation has been termed as irregularly atrial rhythm without clear P waves that was confirmed by a 12-lead ECG. In this study, POAF was defined as any episode of atrial fibrillation lasting more than 5 minutes, with or without symptoms requiring intervention to maintain hemodynamic stability, arisen in the seven days following the cardiac surgery. POAF episodes recorded in condition of hemodynamic and volemic balance were considered for analysis. We excluded POAF episodes potentially related to a sudden fluid loss (diuretic administration, postoperative bleeding, etc..), low blood oxygenation or intravenous high inotropic dose administration.
Anesthesia and surgical technique: Surgical technique and perioperative management were the same for all patients according to the specific surgical procedure. Perioperative anesthesiologic management was the same in all cases: according to the institutional protocol, surgical anesthesia was obtained with continuous intravenous infusion of Propofol + Remifentanil + Cisatracurium, while fluid balance was managed paying attention to the hemodynamic conditions, in order to obtain a mean arterial pressure of at least 70 mmHg. Perioperative drugs management was carried out according to the 2017 EACTS Guidelines on perioperative medication in adult cardiac surgery (14). Before CPB, heparin was intravenously administered at a dose of 300 units/Kg in all cases; protamine need was assessed using the HMS Plus Hemostasis Management system (Medtronic, Minneapolis, MN, USA). Transesophageal echocardiography was routinely performed before the surgical incision in order to assess myocardial and cardiac valves function during and after the surgical procedure. All patients underwent surgery through a standard full sternotomy approach and hypothermic CPB. In order to optimize the surgical times and to avoid confounding factors, all tissue collections were performed before the heparin administration and the placement of extracorporeal circulation cannulas. Surgical excision of EAT was performed from the fat pad of right ventricle infundibulum near the atrioventricular groove, using only a surgical scalpel blade no.11 (without using diathermic) in order to prevent any additional inflammatory damages. After collection, all biopsies were placed in a sterile pipe and quickly transferred to the laboratory to preserve EAT secreting activity. Extracorporeal circulation was performed through aortic and atrio-caval cannulation. All patients requiring high doses of inotropic drugs during their intensive care unit stay were excluded from the study due to the known pro-arrhythmogenic effect (Epinephrine or Norepinephrine > 0,1µg/Kg/min or Dobutamine > 5 µg/Kg/min). At the end of surgery, all patients were moved into the cardiac surgery intensive care unit and weaned from the mechanical ventilation after at least 2 hours of general postoperative monitoring. Fluid intake was regulated to achieve a central venous pressure of at least 10 mmHg according to the cardiac anatomy and myocardial function. All patients received MgSO4 continuous intravenous infusion in a dose of 17,5 gr in the first 24 hours after the surgery as anti-arrhythmia prophylaxis. Packed red cells were transfused only in presence of serum Hemoglobin lower than 8 g/dl while Fresh frozen plasma was used as plasma expander only in case of postoperative bleeding.
Statistical analysis: all statistical analyses were conducted using the statistical platform R (vers. 4.0). Standard descriptive statistics were used to describe the sample: absolute frequencies and percentages for categorical factors and either mean ± standard deviation (std. dev.) or median with range in case of numerical variables. Accordingly, between-groups comparisons were based on the chi-square test (or Fisher exact test where appropriate), the t test for independent samples, or the Mann-Whitney U test. To account for imbalances between the two groups, the inflammatory mediators’ levels were log-transformed and the difference between groups were assessed using a linear model where age, statin use and atrial volume were entered as covariates.
All tests were two-sided with a p value < 0.05 denoting statistical significance. Due to exploratory nature of all the analyses, no adjustments were made for multiple comparison.