Introduction. Transthyretin (TTR) is a plasma protein that transports thyroxin and retinol complexed to retinol-binding protein (RBP). TTR misfolding and aggregation can lead to the extracellular deposition of amyloid in the myocardium, causing TTR cardiac amyloidosis (ATTR-CA). Aim of this study is to develop a native electrophoretic method to characterize circulating TTR forms in plasma samples of ATTR-CA patients, before and after administration of tafamidis, a TTR stabilizer.
Material & Methods. Plasma samples from patients affected by wild-type ATTR amyloidosis (ATTRwt, n=10) were collected before (T0) and during tafamidis treatment at 14, 30 and 90 days. Plasma samples of sex- and age-matched healthy controls (n=6) were also collected. All samples were separated on a native 4–20% Tris-Gly polyacrylamide gel. Western blot analysis was performed with anti-TTR or anti-RBP antibodies. Proteins were detected by Clarity ECL substrate. ImageLab software was used for image acquisition and densitometric analysis of the blots. Total TTR (mg/dl) and RBP (mg/dl) were quantified by nephelometry.
Results. In both groups, the most represented forms were: TTR dimers or trimers (37-50kDa), TTR tetramers complexed with RBP protein in 1:1 (75kDa,) or 1:2 ratio (100kDa), and high molecular weight aggregates (>150kDa). RBP protein was detectable in association with TTR tetramers and some of the TTR aggregates (250kDa, 480kDa). Based on TTR electrophoretic pattern at T0, patients could be divided in 2 groups: ATTR-1, characterised by the presence of TTR tetramers (55kDa), and ATTR-2, characterised by its absence. The ATTR-1 group showed also higher levels of total TTR in comparison with ATTR-2 (mean±SD 25.5±3.8, 16.6±4.2 respectively, p=0.004), while plasma RBP levels were similar (5.1±0.7, 5.1±1.4 respectively). The electrophoretic patterns of ATTR-1 at T0, as well as TTR and RBP levels were comparable to those of controls (TTR 22.6±3.8, RBP 4.4±0.9). Tafamidis treatment was associated with a progressive increase of total TTR levels both in ATTR-1 and -2 (T90 31.4±5.8, 28.7±6.8) but the 2 groups continued to be distinguishable by the electrophoretic pattern.
Conclusions. The native electrophoresis allowed us to detect diverse circulating TTR forms and to classify patients in 2 groups according to their electrophoretic pattern. TTR quali/quantitative evaluation by electrophoresis could represent a valid tool to assess the individual pharmacological response to tafamidis.