Evidence of Specialized Tissue in Human Interatrial Septum: Histological, Immunohistochemical and Ultrastructural Findings

<div><p>Background</p><p>There is a paucity of information on structural organization of muscular bundles in the interatrial septum (IAS). The aim was to investigate histologic and ultrastructural organization of muscular bundles in human IAS, including fossa ovalis (FO) and flap valve.</p><p>Methods</p><p>Macroscopic and light microscopy evaluations of IAS were performed from postmortem studies of 40 patients. Twenty three IAS specimens underwent serial transverse sectioning, and 17 - longitudinal sectioning. The transverse sections from 10 patients were immunolabeled for HCN4, Caveolin3 and Connexin43. IAS specimens from 6 other patients underwent electron microscopy.</p><p>Results</p><p>In all IAS specimens sections the FO, its rims and the flap valve had muscle fibers consisting of working cardiac myocytes. Besides the typical cardiomyocytes there were unusual cells: tortuous and horseshoe-shaped intertangled myocytes, small and large rounded myocytes with pale cytoplasm. The cells were aggregated in a definite structure in 38 (95%) cases, which was surrounded by fibro-fatty tissue. The height of the structure on transverse sections positively correlated with age (P = 0.03) and AF history (P = 0.045). Immunohistochemistry showed positive staining of the cells for HCN4 and Caveolin3. Electron microscopy identified cells with characteristics similar to electrical conduction cells.</p><p>Conclusions</p><p>Specialized conduction cells in human IAS have been identified, specifically in the FO and its flap valve. The cells are aggregated in a structure, which is surrounded by fibrous and fatty tissue. Further investigations are warranted to explore electrophysiological characteristics of this structure.</p></div

Light micrographs of transverse IAS sections.

<p>The heart obtained from a 62-year-old male patient. T-like cells (blue arrowheads), P-like cells (black arrowheads), Purkinje-like cells (turquoise arrowheads). Aggregated T-like cells are surrounded by fibrous tissue (red arrowheads) and then by fat (green arrowheads). End, left atrial endocardium. <b>A.</b> T-like cells, P-like cells, fibrous and fat tissues in the FO (hematoxylin-eosin; ×100). <b>B.</b> The structure consisting of T-like cells surrounded by fibrous and fatty tissue in the FO flap valve (Masson's trichrome; ×100). <b>C.</b> Tortuous, horseshoe-shaped T-like cells forming fan-shaped figures in the FO. (hematoxylin-eosin; ×200). <b>D.</b> T-like cells with side-end contacts (the blue arrowhead) (hematoxylin-eosin; ×400). <b>E.</b> Purkinje-like cells (turquoise arrowheads) are surrounded by T-like cells, fibrous and fatty tissue (hematoxylin-eosin; ×400). <b>F.</b> T-like and P-like cells in the FO flap valve (hematoxylin-eosin; ×1000).</p

An IAS with the flap valve.

<p>The heart obtained from a heart of a 62-year-old female patient. <b>A</b>: A transverse section of the IAS. FO, fossa ovalis; FV, flap valve; LA, left atrium; RA, right atrium. Muscle fibers are shown by purple arrowheads. Black asterisks denote localization of the structure. <b>B, C and D</b>: Light micrographs of consecutive sections of the flap valve; (hematoxylin-eosin; ×50). The sections performed along the flap valve form its basis (A) to the middle part (B), and distally at the central part of the FO. <b>B.</b> Atypical myocytes (specialized-like) are in direct contact with contraction myocardium in the upper part of the interatrial septum. <b>C and D.</b> Along the further course of the structure, it is surrounded by fibrous and fatty tissue. *Typical working cardiomyocytes; †atypical specialized-like myocytes; End, endocardium; fibrous tissue is marked by red arrowheads, fatty tissue is marked by green arrowheads.</p

Left atrial endocardial surface with an IAS and a part of the mitral valve.

<p>The heart obtained from a 55-year-old male patient. FO, fossa ovalis; FV, flap valve; RSVP, right superior pulmonary vein ostium; RIPV, right inferior pulmonary vein ostium; LSVP, left superior pulmonary vein ostium; LIVP, left inferior pulmonary vein ostium; MV, mitral valve. The distance between the RSPV and the flap valve is marked by the green arrow. The dotted oval denotes the most frequently encountered location of the structure.</p

Electron micrographs of P-like cells in the IAS.

<p>The heart obtained from a 55-year-old male patient. P, P-like cell; PC, Purkinje-like cell; Tc, T-like cell; pm, plasma membrane; PV, pinocytic vesicles; bm, basal membrane; m, mitochondria; n, nucleus; pm, plasma membrane; mf, myofilaments; id – intercalated disc; Z, Z-discs. <b>A.</b> A round-shaped P-like cell neighboring a T-like cell and a Purkinje-like cell. <b>B.</b> Plasma membrane of a P-like cell forming multiple pinocytic vesicles and surrounded by the basal membrane. <b>C.</b> A P-like cell with numerous bundles of short myofilament which were cut at various angles. <b>D.</b> A cluster of two P-like cells joined via a complex-shaped intercalated disc.</p

Electron micrographs of Purkinje-like cells in the IAS.

<p>The heart obtained from the same patient as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113343#pone-0113343-g006" target="_blank">figure 6</a>. Pc, Purkinje-like cell; n, nucleus; m, mitochondria; mf, myofilaments; pm, plasma membrane; rer - rough endoplasmic reticulum. <b>A.</b> A transverse section of several Purkinje-like cells. The large size of cells and localization of myofilaments at the cell periphery are appreciated. <b>B.</b> A longitudinal section of a Purkinje-like cell. The regular sarcomeric organization of myofilaments and plasma membrane undulations are visible. A T-like cell is also seen to the left from the Purkinje-like cell. <b>C.</b> Densely packed rough endoplasmic reticulum in Purkinje-like cell cytoplasm.</p

Characteristics of the patients and histological material.

<p>AF, atrial fibrillation; CAD, coronary artery disease; GI, gastrointestinal; COPD, chronic obstructive pulmonary disease; DCM, dilated cardiomyopathy; LV, left ventricle. P>0.05 for all parameters.</p><p>Characteristics of the patients and histological material.</p

Electron micrographs of T-like cells in the IAS.

<p>The heart obtained from a 63-year-old female patient. Tc, T-like cell; mf, myofilaments; Z, Z-discs; m, mitochondria; n, nucleus; pm, plasma membrane; mf, myofilaments; fp – finger-shaped protrusions; id – intercalated disc; gj – gap junction. <b>A.</b> A general view of a T-like cell on the longitudinal section. <b>B.</b> Two bended apposed T-cells. Tc1, Tc2 – two T-cells, arrows indicate a boundary between two T-like cells. <b>C.</b> Bifurcation (arrows) of a T-cell body. <b>D and E.</b> Regular finger-shaped protrusions of the T-cell plasma membrane. <b>F.</b> An intercalated disc between two T-cells (“end to end” contact). <b>G.</b> A gap junction (nexus) between two T-cells (a “side-by-side” contact).</p

A 3-dimensional reconstruction of a specimen containing an IAS.

<p>The reconstruction performed from 107 serial sections of the IAS (obtained from a 50-year old male patient) demonstrates spatial orientation of the structure with the specialized-like myocytes. <b>A:</b> A model of the IAS transverse section (shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113343#pone-0113343-g002" target="_blank"><b>Figure 2A</b></a>). <b>B:</b> A model of the IAS specimen, view from the left atrial side. Red color represents the structure with the specialized-like myocytes. Yellow color represents the fossa ovalis (FO). Green color represents the flap valve (FV). LA, left atrial side; RA, right atrial side.</p