Mechanisms of transthyretin cardiomyocyte toxicity inhibition by resveratrol analogs
Highlights
► We have established a human cardiac tissue culture model system to study the transthyretin cardiac amyloidoses. ► Amyloidogenic transthyretin variants, but not a stable and non-amyloidogenic variant, decrease cardiac cell metabolic activity. ► Resveratrol analogs prevent amyloidogenic transthyretin-induced cardiotoxicity. ► Resveratrol analogs stabilize the transthyretin tetrameric native state and modify the quaternary structure of the monomeric state.
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.
Section snippets
Preparation of recombinant TTR
The proteins were produced in an Escherichia coli expression system [16], [17] and purified at 4 °C, unless stated otherwise. The identity of the proteins was confirmed by liquid chromatography/mass spectrometry.
Cell culture
AC16 cells were grown in DMEM/F12 (1:1) (Cellgro) supplemented with 10% FBS, 2 mM l-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator.
Cell metabolic activity assays
AC16 cells (70–90% confluent) were seeded in black wall clear bottom 96 well plates (250 cells/well) in Opti-MEM,
Human cardiomyocytes are sensitive to TTR variants that deposit in human heart
Human cardiac AC16 cells were treated with several recombinant TTR variants at concentrations ranging from 2 to 16 μM (normal human TTR plasma concentration is 3–7 μM) for 24 h and cell metabolic activity was measured by resazurin reduction assay [18]. TTR variants associated with FAC (V122I TTR, V30M TTR, V20I TTR and L111M TTR) [10], [11], [20], [21] were toxic to the cardiomyocytes in a concentration-dependent manner, whereas the T119M TTR variant, which is stable and non-amyloidogenic [22],
Discussion
While it seems clear that amyloid TTR deposition in peripheral nerves and hearts plays a mechanical role in reducing organ function [29], recent data in cell systems, transgenic mice and human biopsies of peripheral nerves suggest that oligomeric intermediates, which may or may not be on pathway to amyloid fibril formation, produce tissue damage well before tissue deposition can be observed [19], [30], [31]. Furthermore, autopsies from patients carrying the V122I TTR mutation show myocyte
Funding
This work was supported by the American Heart Association (Award 0865061F to N.R.). Additional support from the National Institutes of Health (AG030027 to J.N.B.) is acknowledged. The synthesis of the stilbenes used in this work was supported by the NIH (DK46335 to J.W.K). S.B. is a recipient of a postdoctoral fellowship from the Fonds de la Recherche en Santé du Québec (FRSQ).
Acknowledgment
We thank Dr. Mercy Davidson for developing and providing the human cardiac AC16 cells, and Charles Kacir for technical assistance.
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Present address: Department of New Drug Discovery and Development, Chungnam National University, Daejon 305-764, Republic of Korea.