Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs

Abstract

The transthyretin amyloidoses are a subset of protein misfolding diseases characterized by the extracellular deposition of aggregates derived from the plasma homotetrameric protein transthyretin (TTR) in peripheral nerves and the heart. We have established a robust disease-relevant human cardiac tissue culture system to explore the cytotoxic effects of amyloidogenic TTR variants. We have employed this cardiac amyloidosis tissue culture model to screen 23 resveratrol analogs as inhibitors of amyloidogenic TTR-induced cytotoxicity and to investigate their mechanisms of protection. Resveratrol and its analogs kinetically stabilize the native tetramer preventing the formation of cytotoxic species. In addition, we demonstrate that resveratrol can accelerate the formation of soluble non-toxic aggregates and that the resveratrol analogs tested can bring together monomeric TTR subunits to form non-toxic native tetrameric TTR.

Introduction

The systemic amyloidoses, characterized by the extracellular deposition of amorphous aggregates and amyloid fibrils derived from normal or mutant secreted proteins, are prototypic examples of protein misfolding disorders [1], [2], [3]. Transthyretin (TTR) is one of the 29 human proteins identified thus far, associated with systemic amyloidosis.

TTR is a 55 kDa homotetrameric protein that transports retinol and thyroxine (T₄) in plasma and T₄ in the cerebrospinal fluid. The capacity of TTR to bind a large variety of small molecules [4], other proteins and peptides like the Alzheimer’s disease related Aβ peptide [5], suggests that it may perform other biological functions, perhaps related to detoxification of unwanted metabolites and misfolded proteins [6].

The disassembly of the native homotetrameric TTR to its constituent monomers is required for aggregation and subsequent fibril formation in vitro and presumably in vivo [7]. TTR fibrillogenesis is accelerated by the presence of any of the approximately 100 different amyloidogenic mutations [8], [9], [10], [11] which decrease the thermodynamic and/or kinetic stability of the mutant TTRs with respect to the wild type protein [8].

The clinical syndromes associated with TTR aggregation are senile systemic amyloidosis (SSA), characterized by deposition of wild type TTR (WT TTR) in the heart, and familial amyloidotic polyneuropathy (FAP) and cardiomyopathy (FAC) related to deposition of mutant forms of TTR in the peripheral nerves and the heart, respectively. The V122I TTR variant, found in 3–4% of African-Americans, is the most common amyloidogenic mutation worldwide and it is associated with FAC [11].

Herein, we introduce the human cardiac AC16 cell line as a robust tissue culture system to serve as a model of the TTR cardiomyopathies [12]. AC16 cells are derived from adult ventricular cardiomyocytes, the site of cardiac TTR deposition in SSA and FAC. They express primary cardiomyocyte biochemical markers like α- and β-myosin heavy chain, α-cardiac actin, troponin I, the gap junction proteins connexin-43 and -40, etc., which make them a relevant model for cardiac-specific tissue culture studies [12].

In the present study, we show the effects of several amyloidogenic cardiotoxic TTR variants on cell metabolic activity, which are in stark contrast to the effects of a non-amyloidogenic, stable TTR variant. We used this model to evaluate 22 analogs of the plant polyphenol resveratrol (1) for their capacity to prevent TTR-induced cardiotoxicity. Most of the compounds selected for the screening are potent inhibitors of TTR aggregation and fibril formation in an acid-mediated in vitro assay [13], [14], [15]. We explore the correlation between TTR kinetic stabilization by these compounds and their capacity to prevent cell damage. We also demonstrate that these compounds may inhibit TTR-induced cytotoxicity by more than one mechanism.