Microenvironmental Transthyretin Amyloid Fibrils Alter Cardiac Fibroblast Structure and Function

According to the official reports of the American Heart Association, cardiac diseases are and have been the leading cause of death in the United States during recent years. One disease is infiltrative cardiomyopathy with deposition of abnormal material in the heart tissue, which alters the physiological function and structure of the ventricular walls in one quarter of elderly men and women. However, this wild type transthyretin amyloidosis (wtATTR) is an underappreciated cause of this pathological condition. Animal models for this amyloid disease do not exist, thus in this study, bioengineering approaches have been used to design an in vitro model for this disease, which was exploited to evaluate the main effects and the response at the cellular level to the presence of amyloid TTR. The model is composed of neonatal rat ventricular fibroblasts (NRVFs) cultured on soft polyacrylamide (PA) gel, with stiffness of 10 kPa and stiff glass or plastic (> 1 GPa) coated with a deposited layer of transthyretin amyloid fibrils. Different protein expression and distribution, along with proliferation, adhesion and migration of NRVFs have been qualitatively and quantitatively evaluated with or without the presence of TTR using substrates of different stiffness. To date, there are no studies reporting the behavior of isolated fibroblasts when exposed to TTR fibrils making my study the first. My most significant novel findings for cardiac fibroblasts grown in culture on deposited TTR fibrils are (1) enhanced proliferation rate, (2) the increased migration velocity and (3) the reorganization of the actin cytoskeleton. (1) Increased reduction of MTS tetrazolium by a colorimetric assay suggests a TTR-related increase in the proliferation rate of cells. (2) Barrier removal tests after confluence and single cell live imaging resulted in faster migration velocity of fibroblasts cultured on TTR fibrils. Indeed, two independent methods have been used to evaluate the migration velocity of these cells, both resulting in a faster velocity on TTR-coated substrates. (3) The organization of the actin cytoskeleton within the cells depended on the presence of TTR, in fact actin stress fibers presented a less polarized orientation and a preferential distribution to the borders of the cell when cultured on TTR. I conclude that many of the fibroblast features have been modified by TTR, in some cases very significantly while in others less, both at the structural and functional level. Further studies using different analytical approaches might help in deepening the comprehension of the causes and the mechanisms underlying the pathological adaptation of cells to transthyretin, opening pathways for the development of more realistic in vitro models and subsequently to the discovery of new therapeutic targets, diagnostic instruments and prevention techniques.