Intercalated Disk Extracellular Nanodomain Expansion in Patients With Atrial Fibrillation

Abstract

Aims: Atrial fibrillation (AF) is the most common sustained arrhythmia. Previous evidence in animal models suggests that the gap junction (GJ) adjacent nanodomain – perinexus – is a site capable of independent intercellular communication via ephaptic transmission. Perinexal expansion is associated with slowed conduction and increased ventricular arrhythmias in animal models, but has not been studied in human tissue. The purpose of this study was to characterize the perinexus in humans and determine if perinexal expansion associates with AF.Methods: Atrial appendages from 39 patients (pts) undergoing cardiac surgery were fixed for immunofluorescence and transmission electron microscopy (TEM). Intercalated disk distribution of the cardiac sodium channel Nav1.5, its β1 subunit, and connexin43 (C×43) was determined by confocal immunofluorescence. Perinexal width (Wp) from TEM was manually segmented by two blinded observers using ImageJ software.Results: Nav1.5, β1, and C×43 are co-adjacent within intercalated disks of human atria, consistent with perinexal protein distributions in ventricular tissue of other species. TEM revealed that the GJ adjacent intermembrane separation in an individual perinexus does not change at distances greater than 30 nm from the GJ edge. Importantly, Wp is significantly wider in patients with a history of AF than in patients with no history of AF by approximately 3 nm, and Wp correlates with age (R = 0.7, p < 0.05).Conclusion: Human atrial myocytes have voltage-gated sodium channels in a dynamic intercellular cleft adjacent to GJs that is consistent with previous descriptions of the perinexus. Further, perinexal width is greater in patients with AF undergoing cardiac surgery than in those without.

Quantifying Intermembrane Distances with Serial Image Dilations

“…The experienced user found that W p was significantly wider in patients with AF than without AF (21.9±2.5 and 18.4±2.0 nm, respectively, Figure 9 A ). These values with the correction factor applied are similar to those reported previously (24.4±2.2 nm and 20.7±2.4 nm, respectively) 6 . Importantly, the inexperienced user found the same significant difference (22.1±2.8 nm and 20.1±2.6 nm, respectively) between disease states with the automated program.…”

“…The quantification of the GJ-adjacent perinexus nanodomain width (W p ) is typically accomplished by manual segmentation. This manual segmentation process is demonstrated in Figure 1 A and was described previously 6 . The observer identifies the edge of the GJ ( Figure 1 , red dot), measures 5nm along the center of the perinexus, and measures the distance between the membranes at that point.…”

“…1 and Obs. 2, respectively) - analyzed the same images from a previous study 6 that included 12 patients who were diagnosed with atrial fibrillation (AF) prior to tissue collection and 29 patients that did not have pre-existing AF (No-AF). The experienced user found that W p was significantly wider in patients with AF than without AF (21.9±2.5 and 18.4±2.0 nm, respectively, Figure 9 A ).…”

“…The following algorithm was developed to measure the intermembrane distance between two structurally coupled cardiomyocytes at the point they separate from each other at the edge of a gap junction plaque in a nanodomain called the perinexus 1 , which has been implicated in ephaptic coupling 2 3 4 5 . In the process of analyzing hundreds of transmission electron microscopy (TEM) images of perinexi using a manual segmentation method in a previous study 6 , the need was identified for a higher-throughput method that sampled perinexal width in higher spatial resolution while preserving the accuracy of the previous manual segmentation process During manual segmentation, lines are drawn at 15 nm intervals, approximately orthogonal to the centerline, in order to measure perinexal width.The new algorithm takes a one pixel thick binary outline of two parallel lines and uses serial image dilations to count the number of pixels between the two membranes.While image dilations have commonly been used in a myriad of image processing applications, including contour or edge detection 7 8 , this algorithm uses dilations as a counting mechanism. The centerline is then isolated using a pathfinding algorithm 9 and perinexal width is then measured at a resolution along the length of perinexus equal to the resolution of the image.…”

Cardiac Arrhythmias as Manifestations of Nanopathies: An Emerging View

“…Additionally, in long QT syndrome type 3 (LQT3; a disorder stemming from pathological gain of Na V 1.5 function), the Poelzing group recently demonstrated that transient depletion of extracellular Na + within perinexal nanodomains may mitigate risk of premature beats, and that perinexal widening may unmask the latent arrhythmia risk ( Greer-Short et al, 2017 ). More recently, they have demonstrated that wider perinexi associated with the occurrence of AF in human patients ( Raisch et al, 2018 ). While these are early days yet, these results collectively support the view that ultrastructural alterations within Na V 1.5-rich nanodomains may be a key determinant of arrhythmia risk, and therefore, a potential target for antiarrhythmic therapy.…”