Biochemical and Electrophysiological Modification of Amyloid Transthyretin on Cardiomyocytes

Abstract

Transthyretin (TTR) amyloidoses are familial or sporadic degenerative conditions that often feature heavy cardiac involvement. Presently, no effective pharmacological therapy for TTR amyloidoses is available, mostly due to a substantial lack of knowledge about both the molecular mechanisms of TTR aggregation in tissue and the ensuing functional and viability modifications that occur in aggregate-exposed cells. TTR amyloidoses are of particular interest regarding the relation between functional and viability impairment in aggregate-exposed excitable cells such as peripheral neurons and cardiomyocytes. In particular, the latter cells provide an opportunity to investigate in parallel the electrophysiological and biochemical modifications that take place when the cells are exposed for various lengths of time to variously aggregated wild-type TTR, a condition that characterizes senile systemic amyloidosis. In this study, we investigated biochemical and electrophysiological modifications in cardiomyocytes exposed to amyloid oligomers or fibrils of wild-type TTR or to its T4-stabilized form, which resists tetramer disassembly, misfolding, and aggregation. Amyloid TTR cytotoxicity results in mitochondrial potential modification, oxidative stress, deregulation of cytoplasmic Ca 2þ levels, and Ca 2þ cycling. The altered intracellular Ca 2þ cycling causes a prolongation of the action potential, as determined by whole-cell recordings of action potentials on isolated mouse ventricular myocytes, which may contribute to the development of cellular arrhythmias and conduction alterations often seen in patients with TTR amyloidosis. Our data add information about the biochemical, functional, and viability alterations that occur in cardiomyocytes exposed to aggregated TTR, and provide clues as to the molecular and physiological basis of heart dysfunction in sporadic senile systemic amyloidosis and familial amyloid cardiomyopathy forms of TTR amyloidoses.

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

“…The effects were traced back to modifications in lipid bilayer fluidity/compactness and in cell metabolic/phosphorylation status. These modifications were correlated with cell viability associated with ROS and free Ca 2+ levels 11 . FTIR microspectroscopy is a powerful approach to investigate in situ the modifications occurring in intact cells exposed to harmless or toxic protein aggregates, as well as to their natively folded monomeric counterparts.…”

“…Initially, 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 (pH 7), oligomeric- (OL) or fibrillar-like (FB) aggregates, previously characterized (Figs 1 , S1 , S2 ) 10 , 11 . Figure 1a shows that, after 30 min and 24 h of exposure, the late FB aggregates of both TTR-WT and TTR-L55P were cytotoxic and induced a significant increase of ROS production.…”

“…The observed protein aggregation might therefore be due to the induced stress to endoplasmic reticulum (ER) that could activate the unfolded protein response 48 . Indeed, similarly to the T119M variant natively folded TTR-WT can also be internalized in a subcellular environment 11 . In chicken oocytes, TTR internalization has been described to occur through a clathrin-dependent pathway mediated by a specific receptor 49 .…”

“…When the cells were treated with the L55P variant, not only the amyloidogenic conformers resulted toxic but also the natively folded mutant at pH 7.0, in agreement with its amyloidogenic propensity. The observed toxicity was related, at least in part, to the increased levels of pro-oxidant metabolites that trigger oxidative stress, together with altered Ca 2+ homeostasis 11 . We also found increased autophagic vacuoles in cells exposed for 24 h to TTR-L55P at pH 7.0, while no significant changes were observed when the cells were treated with early or late amyloid aggregates with respect to control cells.…”

“…In our case, the small amounts of oligomers found in the sample of the amyloidogenic variant at pH 7.0 could enhance autophagosome generation to facilitate aggregate removal from the cells. On the contrary, cell treatment with the oligomeric- or fibrillar-like samples by altering Ca 2+ homeostasis and energy metabolism 11 could impair the energy-driven autophagic machinery leading to accumulation of aggregates, oxidative stress and apoptotic cell death.…”