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

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

Determination of Conduction Velocity and Activation delay.

<p><b>A.</b> The ventricles were stimulated from the center of the grid at S₁S₁ stimulation of 100 ms. The stimulation protocol was composed of sixteen basic stimuli followed by 1 premature stimulus (S₁S₂). The premature stimulus started at 90 ms and at the subsequent trains, the coupling interval of the premature stimulus was reduced in steps of 5 ms, until the effective refractory period (ERP) was reached, which was defined as the longest possible coupling interval of the premature stimulus that fails to activate the entire heart. <b>B.</b> CV parallel (longitudinal; CV_L) and perpendicular (transverse; CV_T) to myocyte fiber direction was determined from each activation map. For CVL, the distance between 4 consecutive electrodes parallel to fiber orientation and perpendicular to the isochrones was measured (x) and divided by the time difference (6−2 = 4 ms). Similarly, CVT was determined as Δy/Δt. Activation delay is defined as the local activation time (stimulus is time zero) at a fixed distance from the center of activation origin (stimulus site). Activation delay is registered at two sites (A and B), located on a line parallel to longitudinal and transversal conduction propagation during S₁S₁. Subsequently, activation delay is measured at the same sites during the premature stimuli S₁S₂. Finally, activation delay is normalized by substracting the activation delays of S₁S₁ from activation delay at S₁S₂.</p

Activation maps of wild-type and <i>Scn5a</i> heterozygous RV.

<p>RV activation maps of WT (panel A) and <i>Scn5a</i> heterozygous (panel B) mice paced at S₁S₁ of 100 ms and during S₁S₂ activation with 5 ms decrement until the effective refractory period is reached. Isochronal lines are set to 1 ms. Red denotes earliest activation, blue latest. Equal color represents equal activation times.</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

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

Summary of right and left ventricular CV restitution and activation delay.

<p>k = increased; l = decreased; ∼ = unchanged.</p

Reduced intercellular coupling group conduction velocity and stimulus-to-activation delay measurements.

<p>All values are mean±SEM. S1S2 coupling interval is in ms. CVL/T – longitudinal/transverse conduction velocity (cm/s); StADL/T – stimulus-to-activation delay longitudinal/transverse (ms).</p><p>*intra-variable differences: P<0.05 between consecutive S1S2 and S1S2-5 ms.</p><p>Inter-variable differences are within either RV or LV:</p>§<p>P<0.05 between 100% and 50% Cx43.</p>†<p>P<0.05 between ventricles with 50% and 10% Cx43.</p>¥<p>P<0.05 between ventricles with 100% and 10% Cx43.</p