A novel 3D in vitro model of cardiac transthyretin amyloidosis to investigate amyloid kinetics
S Ministrini, C Balbi, S C Conace, H J Beer, T F Luescher, S F StaempfliAbstract
Background
Wild-type transthyretin amyloidosis (ATTR) is the most common form of transthyretin cardiac amyloidosis [1]. The accumulation of misfolded transthyretin is the result of a complex protein kinetics, involving destabilization, misfolding, deposition and clearance of ATTR [2]; [3]. However, the molecular mechanisms underlying these processes are largely unknown, because no existing disease model is able to provide a comprehensive picture of this complex kinetics [4].
Purpose
to investigate the kinetics of ATTR amyloid in a 3D in vitro model of human heart tissue.
Methods
Myocardial human microtissues (hMTs) were formed by combining defined ratios (4.5:1:1) of human induced pluripotent stem cells derived-cardiomyocytes (hCM), human primary Cardiac Fibroblast (hCF) and human aortic endothelial cells (HAECs), as previously described [5]. hMTs were cultured in endothelial growth medium with 5% fetal bovine serum (FBS), until they create a stable aggregate. Mature hMTs were then incubated in medium with 0.5% FBS, 5% plasma from healthy subjects, or increasing concentrations of plasma from patients with wtATTR cardiomyopathy (1%, 5% and 10%). To dissect the contribution of each cell type to the process of ATTR deposition, they were individually cultured in appropriate conditions and treated as described above. The accumulation of transthyretin was assessed by immunofluorescence, while cell damage was assessed by morphological analysis, and lactic dehydrogenase (LDH) and Troponin T (TnT) release in the medium.
Results
hMTs treated with plasma from patients with wild-type ATTR displayed unspecific signs of cellular injury, like morphological changes, volume reduction and an increased release of LDH and TnT in the medium. This unspecific damage was associated with a concentration-dependent increase of relative TTR content, normalized to total cellularity (assessed by DAPI staining) and cardiomyocytes content (assessed by TnT staining, Figure 1). Interestingly, hMTs incubated with plasma of patients with wild-type ATTR display extracellular accumulation of transthyretin. When analyzing each cell type separately, no significant accumulation of TTR, either intracellular or extracellular, is observed in fibroblasts or cardiomyocytes. Furthermore, they did not show any sign of cell injury. A significant increase in intracellular content of transthyretin is observed instead in endothelial cells, without any sign of cell injury.
Conclusions
these results show for the first time that transthyretin is actively up-taken by endothelial cells before the deposition of ATTR in the extracellular space (Figure 2). Therefore, endothelial internalization could be a limiting step in the process of amyloidogenesis, representing a potential source of inter-individual variability the disease phenotype and a novel potential pharmacological target.Figure 2Figure 1