Cardiac Myocyte and Fibroblast Structure and Function is Affected by Transthyretin Amyloid Deposition

Age-related wild type transthyretin amyloidosis (wtATTR) is characterized by systemic deposition of amyloidogenic fibrils of misfolded transthyretin (TTR) in the connective tissue of many organs. In the heart this leads to broad cardiac dysfunction and is increasingly understood to be a significant cause of age-related heart failure with preserved ejection fraction (HFpEF). The first hypothesis tested is that underlying TTR fibrils disrupt cardiac fibroblast homeostasis which may contribute to fibrosis progression. Primary cardiac fibroblasts had altered cytoskeletal and nuclear architecture when cultured on TTR fibrils, with decreased number of focal adhesions and lower attachment to fabricated micropost substrata. Additionally, deposition of TTR caused cardiac fibroblasts to have increased rates of migration and proliferation. Further, RNA-seq and proteomic analysis showed that cardiac fibroblasts cultured on TTR fibrils had upregulation of immune-related pathways and secretion of proinflammatory cytokines. Taken together, data indicates that TTR fibril deposition alters cardiac fibroblast behavior for immune cell recruitment, along with increased rates of migration and proliferation, which are hallmarks of a fibrosis-promoting fibroblast phenotype. The second hypothesis tested is that TTR deposited in the in vitro model disrupts cardiac myocyte calcium handling, the contraction profile, sarcomere architecture, and cell adhesion complexes. Neonatal rat ventricular myocytes (NRVMs) and adult mouse atrial myocytes exhibited prolonged calcium transients when cultured on TTR fibrils. Additionally, NRVMs cultured on TTR fibrils had decreased expression of the gap junction protein connexin 43 and significantly impaired intercellular connectivity as determined by FRAP. TTR deposition also led to decreased expression of mechanical junction proteins N-cadherin and vinculin. Both NRVMs and human iPSC-derived cardiomyocytes, when cultured on soft hydrogels covered by TTR fibrils, had decreased contractile parameters and force production as observed by traction force microscopy. Finally, NRVMs grown on TTR fibrils had loss of sarcomeric α-actinin with a corresponding increase of ubiquitin localization to the sarcomere. As therapies for wtATTR are cost-prohibitive and only slow disease progression, better understanding of cellular maladaptation in the presence of TTR may elucidate novel therapeutic targets.