Impact of mechanical stress on human transthyretin protein amyloid formation.

Transthyretin (TTR), a human blood circulating protein, undergoes misfolding and fibrillation, leading to transthyretin amyloidosis (ATTR), such as amyloid cardiomyopathy (ATTR-CM) and polyneuropathy (ATTR-PN). While previous studies induced TTR fibrillation under acidic conditions, the role of mechanical shear stress raising from blood flow remains unclear. Herein, we explored how circular shear force affects TTR protein fibrillation using peristaltic pump as an in vitro model mimicking blood flow in pH-neutral buffer. We discovered that TTR mutant proteins causing ATTR-CM (e.g. V122I and WT) were more susceptible to shear force induced fibrillization than ATTR-PN mutant proteins (V30M). In contrast, such differential fibrillation propensity was not observed under the conventional acidic conditions measured by the turbidity assay. Transmission electron microscopy (TEM) revealed distinct fibril morphologies. The impact of mechanical stress on TTR protein fibrillization kinetics was examined according Poiseuille's law. Notably, shear-induced fibrillation was specific to circulation and less obvious under stirring or sonication conditions. Finally, the commercial kinetic stabilizers of TTR proteins Tafamidis and Diflunisal effectively inhibited fibrillation under shear stress. Together, our results highlight ATTR-CM mutants are sensitive to shear force mimicking blood circulation, implying differential etiologies between ATTR-CM and ATTR-PN.