Colocalization of N-cadherin with Cx43.

<p>Immunofluorescence of tissue at different stages of cardiac development double-labeled with N-cadherin (red) and Cx43 (green). Scale bar indicates 20 µm.</p

Assembly of the Cardiac Intercalated Disk during Pre- and Postnatal Development of the Human Heart

<div><p>Background</p><p>In cardiac muscle, the intercalated disk (ID) at the longitudinal cell-edges of cardiomyocytes provides as a macromolecular infrastructure that integrates mechanical and electrical coupling within the heart. Pathophysiological disturbance in composition of this complex is well known to trigger cardiac arrhythmias and pump failure. The mechanisms underlying assembly of this important cellular domain in human heart is currently unknown.</p><p>Methods</p><p>We collected 18 specimens from individuals that died from non-cardiovascular causes. Age of the specimens ranged from a gestational age of 15 weeks through 11 years postnatal. Immunohistochemical labeling was performed against proteins comprising desmosomes, adherens junctions, the cardiac sodium channel and gap junctions to visualize spatiotemporal alterations in subcellular location of the proteins.</p><p>Results</p><p>Changes in spatiotemporal localization of the adherens junction proteins (N-cadherin and ZO-1) and desmosomal proteins (plakoglobin, desmoplakin and plakophilin-2) were identical in all subsequent ages studied. After an initial period of diffuse and lateral labelling, all proteins were fully localized in the ID at approximately 1 year after birth. Na_v1.5 that composes the cardiac sodium channel and the gap junction protein Cx43 follow a similar pattern but their arrival in the ID is detected at (much) later stages (two years for Na_v1.5 and seven years for Cx43, respectively).</p><p>Conclusion</p><p>Our data on developmental maturation of the ID in human heart indicate that generation of the mechanical junctions at the ID precedes that of the electrical junctions with a significant difference in time. In addition arrival of the electrical junctions (Nav1.5 and Cx43) is not uniform since sodium channels localize much earlier than gap junction channels.</p></div

Schematic summary.

<p>Cartoon showing a schematic summary of developmental changes in subcellular immunolocalization of N-cadherin (left, blue), Na_v1.5 (middle, red) and Cx43 (right, green). Black rectangles represent a cardiomyocyte. As pointed out, immunolocalization in cardiomyocytes of plakophilin-2, desmoplakin and plakoglobin is exactly similar to that of N-cadherin at all timepoints.</p

Spatiotemporal movement of Cx43 towards the intercalated disc.

<p>Double labeling of Cx43 (green) and α-actinin (red) at different stages of cardiac development. Cx43 (green) Cx43 moves from the lateral side of the myocytes towards the IDs. Arrows indicate less intense ID staining of Cx43 at the age of 5 years when compared to the intensity of lateral signals. Scale bar indicates 40 µm.</p

Spatiotemporal organization of N-cadherin.

<p>Double labeling of N-cadherin (green) and α-actinin (red) at different stages of cardiac development. Age of the specimen is indicated in the left lower corner of the panels. Scale bar indicates 20 µm.</p

Spatiotemporal organization of desmosomal proteins.

<p>During all development stages the desmosomal proteins desmoplakin, plakoglobin and plakophilin-2 (green) colocalize with N-cadherin (red). The plakoglobin signals revealed besides a colocalization with N-cadherin at the IDs also a staining of the capillaries (arrows) between the myocytes. Scale bar indicates 20 µm.</p

Age of all cardiac specimens that were studied.

<p>Interval indicates post-mortem period till tissue preservation. m =  male, f =  female.</p

Molecular functions of designated ID proteins.

<p>Subnetwork GO analysis of the 97 candidate proteins observed in the membrane or integral to membrane compartment in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g002" target="_blank">Fig 2</a>. Proteins were clustered according to their GO annotation in process and function. Comparable to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g002" target="_blank">Fig 2</a>, the color of the nodes represents their fold-enrichment measured (ER). The cluster in the middle contains all currently known junction proteins (based on GO and Estigoy et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.ref022" target="_blank">22</a>]). Proteins in this cluster are connected to proteins with other biological functions based on known protein-protein interactions (lines). The four candidate proteins selected for follow-up studies are enlarged.</p

Flotillin-2 shows increased ID expression during cardiac disease.

<p>(A) Immunofluorescence imaging of N-cadherin and flotillin-2 in human left ventricular tissue. The overlay image reveals strong co-localization. (B) Co-localization studies of N-cadherin and flotillin-2 in left ventricular tissue from two patients with DCM hints at an increased level of flotillin-2 at the ID. Intensity and localization of fluorescent signals were analyzed through line scans and are plotted for both patients (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.s002" target="_blank">S2 Fig</a>). Robust co-localization was confirmed through correlation coefficients of R² = 0.81 and 0.86 for the upper panel and lower panel, respectively. (C) Representative Western blots of the CF and EMF in healthy human tissue, compared to left ventricular tissue of patients with either DCM or ARVC. Differences between the levels in CF and EMF were analyzed using an unpaired t-test. Differences between the groups of patients were analyzed via a one-way ANOVA. * P<0.05, ** P<0.005, ***P<0.0005.</p

Molecular context of proteins in the EMF.

<p>Schematic representation of protein-protein interactions and cellular localization across the 366 candidate proteins that showed enrichment (ER>10-fold) in the EMF-fraction. This highly connected network is based on protein-protein interactions currently available in the Biogrid database in mouse, rat and human (lines). The color of the nodes represents the fold-enrichment measured; darker colors mean higher enrichment ratios. All known cell junction proteins, as based on GO-terms and a recent <i>in silico</i> study by Estigoy et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.ref022" target="_blank">22</a>], are visualized by their gene names and enlarged circles. As expected, these mainly clustered in the plasma membrane region and several known ID protein complexes were readily identified at this intracellular location. These and all other (connected) proteins in this compartment were considered as most likely ID candidates and further evaluated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0152231#pone.0152231.g003" target="_blank">Fig 3</a>.</p