Cholesterol Efflux Capacity in Atrial Fibrillation

To assess the association between the pathophysiology in AF and HDL functionality, we measured three types of HDL CECs by using serum of patients with AF who were about to undergo catheter ablation. Global CEC, ABCA1 CEC, and Non-ABCA1 CEC of HDL were measured using methods previously described with modifications^{39 (link)}. Apolipoprotein B (apoB)-depleted serum was prepared by adding 40 μL of 20% polyethylene glycol 6000 (Sigma-Aldrich, St. Louis, MI, USA) in 200 mM glycine (pH 7.4) to 100 μL of serum and then centrifugation at 8,000 × g for 30 min. The supernatants were stored at -80 °C until use. J774.1 murine macrophage-like cells, obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (Osaka, Japan), were seeded in 48-well plates at 75,000 cells per well and then labeled with [³H]-cholesterol (1 μCi/mL, PerkinElmer, Waltham, MA, USA) for 24 h at 37 °C in RPMI1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 5% fetal bovine serum (FBS), acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor Sandoze 58–035 (2 μg/mL, Sigma-Aldrich), and 50 μg/mL acetylated LDL (Ac-LDL, Alfa Aesar, Ward Hill, MA, USA) to enrich the cells with free cholesterol. The cells were equilibrated for 24 h in RPMI1640 medium supplemented with 0.2% bovine serum albumin (Roche, Basel, Switzerland) and 2 μg/mL ACAT inhibitor in the presence (Global CEC) or absence (Non-ATP-binding cassette transporter A1 (ABCA1 CEC) of cyclic-3′,5′-AMP (CTP-cAMP, Cayman Chemical Company, Ann Arbor, MI, USA) to induce ABCA1 expression. Efflux media containing 2.8% apoB-depleted serum was added for 4 h. The CEC of HDL was calculated as the percentage of radioactivity recovered from the medium to total radioactivity count. Three types of CECs were finally normalized with the CEC value of pooled control serum collected from healthy volunteers (age range, 27–32 years old, n = 4) on each plate to be presented as CEC indexes. ABCA1 CEC was calculated by subtracting the Non-ABCA1 CEC from Global CEC.