Hypertension is associated with preamyloid oligomers in human atrium: a missing link in atrial pathophysiology?

Abstract

Background: Increasing evidence indicates that proteotoxicity plays a pathophysiologic role in experimental and human cardiomyopathy. In organ-specific amyloidoses, soluble protein oligomers are the primary cytotoxic species in the process of protein aggregation. While isolated atrial amyloidosis can develop with aging, the presence of preamyloid oligomers (PAOs) in atrial tissue has not been previously investigated.

Methods and results: Atrial samples were collected during elective cardiac surgery in patients without a history of atrial arrhythmias, congestive heart failure, cardiomyopathy, or amyloidosis. Immunohistochemistry was performed for PAOs using a conformation-specific antibody, as well as for candidate proteins identified previously in isolated atrial amyloidosis. Using a myocardium-specific marker, the fraction of myocardium colocalizing with PAOs (PAO burden) was quantified (green/red ratio). Atrial samples were obtained from 92 patients, with a mean age of 61.7±13.8 years. Most patients (62%) were male, 23% had diabetes, 72% had hypertension, and 42% had coronary artery disease. A majority (n=62) underwent aortic valve replacement, with fewer undergoing coronary artery bypass grafting (n=34) or mitral valve replacement/repair (n=24). Immunostaining detected intracellular PAOs in a majority of atrial samples, with a heterogeneous distribution throughout the myocardium. Mean green/red ratio value for the samples was 0.11±0.1 (range 0.03 to 0.77), with a value ≥0.05 in 74 patients. Atrial natriuretic peptide colocalized with PAOs in myocardium, whereas transthyretin was located in the interstitium. Adjusting for multiple covariates, PAO burden was independently associated with the presence of hypertension.

Conclusion: PAOs are frequently detected in human atrium, where their presence is associated with clinical hypertension.

Trial registration: ClinicalTrials.gov NCT00141778.

Keywords: amyloid; atrial natriuretic peptide; atrium; preamyloid oligomers.

© 2014 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.

Figures

Figure 1.

Distribution of preamyloid oligomers (PAOs) in human atrium. Representative human atrial samples with a low (sample 1), medium (sample 2), and high (sample 3) green/red ratio (G/R) value are shown. Immunolabeling results with both myosin heavy chain‐specific monoclonal antibody (MF‐20) and PAO‐specific antibodies (A‐11; Column A), or A‐11 alone (Column B). Column C. Background fluorescent signal after excitation using 488 nm in the presence of nonspecific IgG. Columns D, E. Images after creation of the myocardium binary mask and PAO signal within the myocardium (with G/R value), respectively. Scale bars=50 μm.

Figure 2.

Median and individual green/red ratio (G/R) values for all patient samples. For each patient sample (x axis), the range (vertical bars), median (black horizontal bars), and SD (dotted lines) of the individual imaging field G/R values (y axis) are illustrated. Whiskers are absent for data sets in which the 25% or 75% quantiles are equal or close to the median value. Data for patients with hypertension are shown in red, while blue indicates no history of hypertension.

Figure 3.

Representative histologic features in atrial samples from patients with low, medium, and high green/red ratio (G/R) values. For each patient sample (rows), the columns from left to right display images showing: hematoxylin and eosin–stained section (A); Congo red–stained section with visualization using polarized light (B); and Masson's trichrome–stained section demonstrating collagen (blue) representing areas of fibrosis (C). The G/R values are shown to the left of the images.

Figure 4.

Colocalization of ANP and PAO immunoreactivity. Immunofluorescent labeling with PAO‐specific (A‐11; Panel A) and ANP‐specific (B) polyclonal antibodies in adjacent 5‐μm human atrial tissue samples. C, Binary mask, representing the area of atrial myocardium. D, Colocalization of A‐11 (green) and ANP (red) signals within the myocardium in adjacent sections, as evidenced by the lighter green and yellow regions, compared with (A). Scale bars=50 μm. ANP indicates atrial natriuretic peptide; PAO, preamyloid oligomers.

Figure 5.

Lack of colocalization for TTR and PAO. Immunofluorescent labeling with PAO‐specific (A‐11; A) and TTR‐specific (B) polyclonal antibodies (TTR) in adjacent 5 μm human atrial tissue samples. C, Binary mask, representing the area of myocardium. D, Resulting merged image of A‐11 (green) and TTR (red) signals in adjacent sections, demonstrating minimal evidence of colocalization. Scale bars=50 μm. PAO indicates preamyloid oligomers; TTR, transport protein transthyretin.

Figure 6.

Hypothesis linking hypertension, PAO formation, and atrial pathology/arrhythmogenesis, compared with the known process of isolated atrial amyloidosis. In the presence of hypertension, protein misfolding and oligomerization can occur in the atrium, with ANP a principal component of PAOs. Protein oligomers can directly cause myocyte injury/death, or they can coalesce with aging to form amyloid fibril deposition in atrial myocardium. This process, known as isolated atrial amyloidosis, is associated with infiltrative structural damage in the atrium and an increased risk of atrial fibrillation. ANP indicates atrial natriuretic peptide; PAO, preamyloid oligomers.