Different factors affecting human ANP amyloid aggregation and their implications in congestive heart failure

Abstract

Aims: Atrial Natriuretic Peptide (ANP)-containing amyloid is frequently found in the elderly heart. No data exist regarding ANP aggregation process and its link to pathologies. Our aims were: i) to experimentally prove the presumptive association of Congestive Heart Failure (CHF) and Isolated Atrial Amyloidosis (IAA); ii) to characterize ANP aggregation, thereby elucidating IAA implication in the CHF pathogenesis.

Methods and results: A significant prevalence (85%) of IAA was immunohistochemically proven ex vivo in biopsies from CHF patients. We investigated in vitro (using Congo Red, Thioflavin T, SDS-PAGE, transmission electron microscopy, infrared spectroscopy) ANP fibrillogenesis, starting from α-ANP as well as the ability of dimeric β-ANP to promote amyloid formation. Different conditions were adopted, including those reproducing β-ANP prevalence in CHF. Our results defined the uncommon rapidity of α-ANP self-assembly at acidic pH supporting the hypothesis that such aggregates constitute the onset of a fibrillization process subsequently proceeding at physiological pH. Interestingly, CHF-like conditions induced the production of the most stable and time-resistant ANP fibrils suggesting that CHF affected people may be prone to develop IAA.

Conclusions: We established a link between IAA and CHF by ex vivo examination and assessed that β-ANP is, in vitro, the seed of ANP fibrils. Our results indicate that β-ANP plays a crucial role in ANP amyloid deposition under physiopathological CHF conditions. Overall, our findings indicate that early IAA-related ANP deposition may occur in CHF and suggest that these latter patients should be monitored for the development of cardiac amyloidosis.

Figure 1. Aggregation kinetics.

A) Aggregation kinetics of α-ANP via Congo red binding method. Data represent the mean of at least three independent experiments (n≥3). Errors bars represent standard deviations B) Glutaraldehyde Cross-linking, Tris-tricine SDS-PAGE and SDS-PAGE at different times. The formation of α-ANP oligomers and high MW aggregates was monitored. Asterisks indicate dimers, arrows indicate monomers and # indicate high molecular weight aggregates. C) Morphology of α-ANP aggregates at different times and in different conditions. α-ANP was applied onto a copper grid and negatively stained with 2 (w/v)% uranyl acetate. Scale bar:100 nm.

Figure 2. Fourier-self deconvoluted Amide I spectral region of α-ANP in aqueous solution.

a: pH 7.4, from the bottom to the top t = 0 h, t = 5 h, t = 30 h, t = 168 h. b: pH = 4.0, from the bottom to the top t = 0 h, t = 1 h, t = 48 h, t = 168 h. Arrows indicate the increase/decrease of the relative band intensities with time.

Figure 3. Seeding.

A) Seed-induced fibril formation of α-ANP . Seeds were generated from aged α-ANP (pH 7.4 and pH 4.0, respectively) and added as percentage weight fractions. Fibril formation was monitored over time by Cb and TEM. B) Treatment in 90% HFIP of ANP aggregates formed after seeding . High MW aggregates from pH 4.0 seeded solution remained on the top of the gel while aggregates formed by pH 7.4 seeds were monomerized. C) Morphology of samples aged for 720 hours. Morphologies of seeded and unseeded α-ANP at pH 7.4 were still similar in shape. D) pH dependence of α-ANP aggregation. α-ANP was incubated at various pH and after two days the amount of aggregation was quantified by Cb.

Figure 4. Time course of ANP-CHF fibril formation.

A) Cb and B) Th-T fluorescence. TEM image after 30 days showed amyloid fibril larger in cross sectional area and longer in length than fibrils formed in all other conditions tested.

Figure 5. TEM images.

Electron micrographs showing the ANP-CHF time course aggregation at pH 7.4 (A,B,C,D) and at pH 7.4 (E,F,G,H).

Figure 6. TEM images.

Electron micrographs showing the α-ANP time course aggregation at pH 4.0 (A,B,C,D) and in 0.5 mM SDS at pH 7.4 (E,F,G,H).

Figure 7. Stability of ANP aggregates via GdnHCl addition.

ANP-CHF fibrils were the most resistant since only above 4 M GdnHCl, an increasingly greater percent of total fibrils was resolubilized.

Figure 8. Theoretical schema of ANP aggregation.