Abstract
Background Transthyretin amyloidosis (ATTR) is a progressive, degenerative disease affecting the heart and other organ systems, as well as the peripheral, autonomic, and central nervous systems. Although pharmacological and genetic evidence establishes aggregation as a driver of ATTR pathology, the mechanism by which aggregation compromises post-mitotic tissue function is poorly understood. We utilized bottom-up proteomics on wild-type (WT) human cardiac (WT/WT genotype) and V122I human cardiac (V122I/WT genotype) tissue, combined with tissue clearing technology to create an optically transparent tissue architecture to visualize three-dimensional relationships, to better understand TTR cardiomyopathy (CM).
Methods Flash-frozen 0.5 mm cardiac tissue slices from human subjects with end-stage WT-TTR CM, end-stage V122I CM, and slices from an age-matched human control were used for these experiments. Fibril extraction from diseased tissue followed published protocols. Strong denaturant-mediated proteome tissue extraction on samples from each subject facilitated bottom-up proteomics by using liquid chromatography (LC)-mass spectrometry (MS)/MS. Tissue clearing was performed on 0.5 mm cardiac slices utilizing a lauryl sulfate-based lipid removal strategy. Slices were stained using indirect immunofluorescence with antibodies to protein targets identified by proteomics. We used an antibody to non-native TTR and AmyTracker 480 (an oligothiophene dye that binds to amyloid fibrils) to image TTR deposits. ATTR fibrils were characterized structurally using cryogenic electron microscopy (cryo-EM) followed by helical reconstruction.
Results Proteomic cardiac analysis afforded high spectral counts for transthyretin (TTR) and proteins typically associated with amyloid fibrils, e.g. serum amyloid P (APCS). Fibril and cardiac homogenate proteomics revealed high levels of angiogenic and hemostatic proteins, including those composing the complement and coagulation cascades. 3D imaging revealed loss of normal microvascular architecture in CM samples with regions of hyper- and hypovascularization. Microvascular obstruction by capillary thrombosis was also observed in CM. ATTR fibrils adopted the common spearhead fold and were decorated with collagen VI (COLVI), an extracellular matrix component.
Conclusions We hypothesize that ATTR CM is a microangiopathy driven by capillary bed thrombo-inflammation and dysregulated angiogenic revascularization. Phenotypic convergence of WT ATTR CM and V122I ATTR CM was observed via proteomics, 3D imaging, and ex vivo fibril characterization by cryo-EM. We provide evidence of capillary thrombosis in ex vivo ATTR CM tissue. Vasodilation and increased capillary permeability expose components of the vascular basement membrane (VBM) to misfolded TTR. These components are known to promote TTR aggregation and stabilize amyloid fibrils in the extracellular space. Congestion of the VBM prevents appropriate revascularization, reducing cardiac exertional capacity over time, leading to heart failure. Our ATTR CM heart tissue proteomics data shows significant overlap with the proteomic profiles of human AD brain tissues, revealing key amyloid, coagulation, complement, and angiogenesis proteins being changed in amyloidoses.
Introduction
Transthyretin amyloidosis (ATTR) is a progressive degenerative disease, often initially affecting the heart and/or the peripheral and autonomic nervous systems, and eventually the central nervous system.1,2 This disease can be inherited (autosomal dominant) or can occur sporadically as an apparent consequence of aging. When principally affecting the heart, if untreated, ATTR cardiomyopathy (CM) progresses to heart failure with preserved ejection fraction and eventually death in approximately 5-7 years.2,3 Pharmacological and genetic evidence support the hypothesis that the post-mitotic tissue degeneration in ATTR is caused by aggregation of the human plasma protein transthyretin (TTR).2,3 Kinetic stabilizers such as tafamidis increase the energetic barrier for TTR tetramer dissociation, the rate-limiting step for TTR aggregation, through native state kinetic stabilization, thereby inhibiting aggregation of TTR.4,5 Tafamidis dosing thus slows down the progression of both hereditary and sporadic WT ATTR cardiomyopathy by inhibiting aggregation of newly biosynthesized TTR.4
While TTR primarily functions as the main carrier of holo-retinol binding protein and as a secondary carrier for thyroxine,6,7 studies have also intimated TTR’s ability to regulate angiogenic functions, including capillary growth and endothelial cell proliferation in lung tumors via Akt.8,9 The TTR tetramer has been shown to increase expression of LRP1,10 a receptor that plays a pivotal role in organized angiogenesis and acts, in part, via Akt.11 LRP1 also has a role in the neuronal internalization of amyloidogenic proteins such as tau12 and α-synuclein,13 as well as participating in TTR-Aβ trafficking,14 indicating a potential role for LRP1 in ATTR development. Interestingly, V30M TTR has been shown to downregulate many pro-angiogenic genes in endothelial cell culture,9 suggesting TTR tetramer stability may play a role in TTR’s effectiveness in regulating angiogenesis. Indeed, mouse models of Alzheimer’s disease treated with TTR tetramer stabilized with iododiflunisal displayed decreased vascular pathology compared to controls.10 Further evidence suggesting a potential role of the native tetrameric structure in TTR’s functional angiogenic properties is seen in the gradual increase in circulating endothelial progenitor cells in cardiac ATTR patients treated with the TTR stabilizer tafamidis.15
The pathophysiology of ATTR CM remains unclear. One hypothesis is that TTR amyloid fibril accumulation in the myocardium leads to a restrictive or infiltrative CM, wherein muscle tissue in the heart’s lower chambers (ventricles) becomes stiff, and the ventricles cannot fill with blood efficiently, leading to reduced flow of blood in the heart.1,16 Histologically, ATTR amyloid deposition has been described as patchy, i.e. without a discernable deposition pattern, in contrast to the perivascular pattern observed in other forms of cardiac amyloidosis, such as in immunoglobulin light chain amyloidosis (AL).1,3 A 2016 histopathological study of both ATTR and AL cardiac amyloidoses described the most common ATTR deposition pattern as discrete pericellular, though nodular and interstitial deposition patterns were also observed.17 While vascular deposition was much more prevalent in AL pathology, the study showed ATTR deposition in the intramyocardial vasculature, especially in intramyocardial arteries.17