Characterizing the effect of transthyretin amyloid on the heart
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Transthyretin (TTR)-associated amyloidoses are diseases wherein wild-type or mutant TTR forms amyloid fibrils that infiltrate multiple organs. Wild-type TTR amyloidosis, ATTRwt, is a sporadic disease characterized by deposits that occur mainly in the heart. Alternatively, >100 TTR mutants cause inherited forms, ATTRm, frequently featuring cardiac amyloid deposits. The goals of this research were to create a cell-based model of ATTR amyloidosis, to define the mechanism of cardiac TTR-associated amyloid at the cellular level, and to study several agents that could interrupt the amyloid process. We hypothesized that TTR oligomers were cardiotoxic and played a role in the mechanism of ATTR amyloidosis, and that cytotoxicity could be inhibited by diflunisal, doxycycline, and Kiacta®. Focusing on TTR proteins associated with cardiac amyloidosis (wild-type, L55P, V30A, and V122), we developed a thermal denaturation method for creating TTR oligomers that allowed us to study the direct effect of oligomers on cells. Congo red and thioflavin T analyses confirmed that the oligomers were on pathway to amyloid fibril formation. We tested the effect of TTR oligomers on rat and human cardiac cells by measuring cell viability and stress response (through live protease activity and qPCR). TTR-L55P oligomers elicited a cytotoxic effect; fluorescent microscopy indicated cellular uptake of the oligomers and continued intra-cellular aggregation. Cytotoxicity was blocked when TTR was heated in the presence of doxycycline; the drug appeared to dissociate TTR aggregates or stabilize the monomeric forms. We also investigated retinol-binding protein (RBP), a natural binding partner of TTR. By immuno-histochemistry, RBP was demonstrated in ATTRwt and ATTRm `non-amyloid' transplant heart tissues, localized to areas containing amyloid or in the case of the transplant tissue, regions that appeared to display ischemic damage. Serum RBP levels were significantly different in ATTR vs. age-matched controls (p = 0.03), and in ATTRwt vs. ATTRm (p <0.0001) by ELISA. These data provide evidence that TTR oligomers are cardiotoxic, possibly due to cellular internalization and progressive intracellular aggregation. Furthermore, our results support the use of doxycycline as a therapeutic in ATTR to target these amyloidogenic oligomers, and suggest that RBP may have potential as a disease biomarker.