Light Chain Amyloid Fibrils Act as a Recruitment Point for Soluble Protein and Reveal their Cytotoxic Potential in Human Cardiomyocytes

Abstract

Light chain (AL) amyloidosis is an incurable human disease characterized by the misfolding and systemic amyloid aggregation of immunoglobulin light chains. While several studies have reported that soluble light chain internalize into renal and cardiac fibroblast cells, the correlation between cellular internalization and the mechanisms causing AL cytotoxicity still remains unclear. Previously, we demonstrated that AL proteins internalize into mouse cardiomyocytes and localize into lysosomes. Here, we extended this study following the internalization of full length (FL) and variable domain (VL) AL proteins into human cardiomyocytes (AC16 cell line) by real-time live-cell image analysis (IncuCyte ZOOM™). Our results show that soluble protein internalizes into AC16 cells in a size-dependent manner, thus VL domains internalizes faster than the FL proteins. Experiments using endocytic inhibitors have shown that soluble proteins internalize by a Clathrin-dependent endocytic pathway and localize in perinuclear compartments. Further on, we have studied the internalization of preformed amyloid fibrils and their effect when co-incubated with soluble protein. External aggregates rapidly surround the cells, strongly interact with the cell membrane, and act as a recruitment point for soluble protein, triggering the amyloid fibril elongation (cell mediated seeding). Although fibrils are predominantly extracellular, a fraction of them is also internalized. Finally, our attempt to clarify the role of amyloid fibrils in the disease revealed their cytotoxic potential at significant low concentrations compared to soluble AL proteins. This study would help us to understand the unique aspects of behind light chain internalization and cytotoxicity in AL amyloidosis.