A FTIR microspectroscopy study of the structural and biochemical perturbations induced by natively folded and aggregated transthyretin in HL-1 cardiomyocytes

Diletta Ami,Antonino Natalello,Manuela Leri,Sofia Giorgetti,Monica Bucciantini,Massimo Stefani,Paolo Mereghetti,Silvia Maria Doglia

doi:10.1038/s41598-018-30995-5

Diletta Ami, Antonino Natalello + Show 6 more

Open Access

https://doi.org/10.1038/s41598-018-30995-5

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Abstract

Protein misfolding and aggregation are associated with a number of human degenerative diseases. In spite of the enormous research efforts to develop effective strategies aimed at interfering with the pathogenic cascades induced by misfolded/aggregated peptides/proteins, the necessary detailed understanding of the molecular bases of amyloid formation and toxicity is still lacking. To this aim, approaches able to provide a global insight in amyloid-mediated physiological alterations are of importance. In this study, we exploited Fourier transform infrared microspectroscopy, supported by multivariate analysis, to investigate in situ the spectral changes occurring in cultured intact HL-1 cardiomyocytes exposed to wild type (WT) or mutant (L55P) transthyretin (TTR) in native, or amyloid conformation. The presence of extracellular deposits of amyloid aggregates of WT or L55P TTR, respectively, is a key hallmark of two pathological conditions, known as senile systemic amyloidosis and familial amyloid polyneuropathy. We found that the major effects, associated with modifications in lipid properties and in the cell metabolic/phosphorylation status, were observed when natively folded WT or L55P TTR was administered to the cells. The effects induced by aggregates of TTR were milder and in some cases displayed a different timing compared to those elicited by the natively folded protein.

Highlights

The pathologic presence of extracellular or intracellular insoluble fibrillar deposits of well identified peptides/ proteins in specific organs and tissues is a shared feature of amyloid diseases
We investigated by Fourier transform infrared (FTIR) microspectroscopy the biochemical modifications occurring in HL-1 cells exposed to wild type TTR (TTR-WT) or to a highly amyloidogenic variant (TTR-L55P) associated with aggressive forms of familial amyloid polyneuropathy (FAP), either as such or at varying aggregation times
We analysed the viability and reactive oxygen species (ROS) production in HL-1 cardiomyocytes exposed for 30 min or 24 h to TTR-WT or to TTR-L55P in different conformational states: native, oligomeric- (OL) or fibrillar-like (FB) aggregates, previously characterized (Figs 1, S1, S2)[10,11]

Summary

Introduction

The pathologic presence of extracellular or intracellular insoluble fibrillar deposits of well identified peptides/ proteins in specific organs and tissues is a shared feature of amyloid diseases. In the case of complex biological systems, such as intact cells, this spectroscopic approach provides, within a single measurement, information on the main biomolecules found in the sample, including lipids, proteins, nucleic acids, and carbohydrates[6,7]. Our study aims at providing novel information on the amyloid toxicity theme by an innovative approach to describe a global cell signature resulting from exposure to natively folded or misfolded/aggregated TTR. To this purpose, different functional groups of macromolecules and their changes induced by externally added misfolded/aggregated TTR were monitored in situ in cultured intact HL-1 cardiomyocytes

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