Proteotoxicity in Cardiac Amyloidosis: Amyloidogenic Light Chains Affect the Levels of Iintracellular Proteins in Human Heart Cells
2017
Abstract Introduction. AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. In addition to the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at the cellular level are complex and largely undefined. Herein we sought to characterize the molecular changes occurring in primary human cells (cardiac fibroblasts, hCF) exposed in vitro to soluble amyloidogenic cardiotropic LCs, and control LC from patients with multiple myeloma, by evaluating their proteome. Methods. Monoclonal LCs were purified from patients with AL amyloidosis and severe heart involvement (CardioLCs), and from multiple myeloma patients without amyloidosis (MMLCs, used as controls). The effects of various LCs (4 CardioLCs and 3 MMLCs) on hCF physiology were explored regarding viability and cytotoxicity. A combination of gel-based and label-free shotgun analyses, followed by bioinformatics and data validation studies, was used to characterize the proteome alterations of cultured hCF exposed for 24h to a representative cardiotropic LC (CardioLC1), by comparison with untreated cells and with cells incubated with a representative MMLC (MMLC2). In order to assess the generalizability of the results, the quantitative changes in a subset of proteins have been further assessed by immunoblotting in hCF exposed to each of the LCs employed in the study. Results. Incubation of hCF with the various cardiotropic LCs translates into impaired viability, with apoptosis activation and oxidative stress. These alterations are associated with significant proteome remodeling: combining the results of the two proteomic methodologies, 85 differentially represented proteins were found overall in the comparison between CardioLC1-treated and untreated hCF, and 51 in the CardioLC1-treated versus the MMLC2-treated comparison. The differential proteins are involved in key cellular processes, including cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. Among the proteins identified as differential by proteomics, 8 species (Talin, Heat shock protein 27, Transgelin, Porin 31HM, Proteasome subunit beta type-2, Cysteine and glycine-rich protein 2, Cullin-associated NEDD8-dissociated protein 1 and Vimentin) have been selected overall for independent validation by western blotting. Selection was based on their fold change, biological role and/or availability of validated antibodies. Changes are being confirmed to occur also in hCF treated with the other cardiotropic LCs. Conclusions. These results support and expand the concept that soluble species of amyloidogenic cardiotropic LCs possess toxicity towards cardiac cells. Overall, our investigation has allowed characterizing the molecular alterations in target cells exposed to an extrinsic proteotoxic stimulus, represented by monoclonal cardiotropic LCs. Quantitative changes in specific proteins may in the future be explored as potential markers of cardiac proteotoxic stress. Dysregulation of species involved in crucial cellular functions as cytoskeletal remodeling, mitochondrial activity and metabolism, protein synthesis, quality control and degradation, may play a causative role in the cellular alterations that lead to the clinical picture, and targeting these altered pathways may be beneficial for counteracting organ damage in AL amyloidosis. Disclosures Palladini: Prothena: Honoraria, Other: Travel grant; Celgene: Other: Travel grants; Jannsen-Cilag: Membership on an entity's Board of Directors or advisory committees.
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