Three‐Dimensional Visualization and Proteomic Analysis of Human Cardiac Transthyretin Amyloidosis Tissue Reveals Microangiopathy and Capillary Occlusion

BACKGROUND: Transthyretin amyloidosis (ATTR) is a degenerative disease affecting the heart and other organs. Transthyretin (TTR) aggregation is a driver of ATTR pathology, but the mechanism is poorly understood. We used proteomics and tissue clearing technology on wild-type (WT) human cardiac (WT/WT) and V122I human cardiac (V122I/WT) tissue to better understand TTR cardiomyopathy. METHODS: Flash-frozen cardiac tissue slices from human subjects with end-stage WT-TTR cardiomyopathy, end-stage V122I TTR cardiomyopathy, and an age-matched control were used. Fibrils and tissue proteomes were extracted and assessed by bottom-up proteomics. Tissue clearing was performed using a lauryl sulfate-based lipid removal strategy. Slices were stained using indirect immunofluorescence against targets identified by proteomics. TTR deposits were imaged by antibody and AmyTracker 480 staining. Structures of ATTR fibrils were characterized using cryogenic electron microscopy. RESULTS: Proteomic analysis revealed high abundance of TTR, proteins associated with amyloid fibrils, as well as angiogenic, hemostatic, and complement cascade-associated proteins. Three-dimensional imaging revealed loss of normal microvascular architecture, regions of hypervascularization and hypovascularization, and microvascular obstruction by capillary thrombosis. ATTR fibrils adopted the spearhead fold and were decorated with collagen VI, an extracellular matrix component. CONCLUSIONS: Based on our imaging and proteomic data, we hypothesize that ATTR cardiomyopathy is a microangiopathy driven by capillary bed thromboinflammation and dysregulated angiogenic revascularization. In this model, increased capillary permeability exposes components of the vascular basement membrane to misfolded TTR. These components promote aggregation and stabilize amyloid fibrils. Congestion of the vascular basement membrane prevents appropriate revascularization, reducing cardiac exertional capacity over time, leading to heart failure.