3D Tomographic Segmentation of Adult Cardiac Ventricle reveals a Complex Tubular and Vesicular Network surrounding the Gap Junction Plaque. Ultrastructure of the Connexome

Abstract

<h3>Background</h3> The cardiac intercalated disc (ID) has been extensively studied by conventional transmission electron microscopy (EM). Yet, novel methods for tissue preservation (high-pressure freezing), image (3D tomographic EM) and analysis (image segmentation) that greatly improve image quality/resolution, have not been applied to the ID. Recent studies show that, at the ID, the gap junction protein Connexin43 is part of an interactome (a "connexome"). Here, we provide a structural characterization of the connexome. <h3>Methods</h3> Adult mouse ventricular tissue was prepared by high-pressure freezing and freeze substitution and embedded in resin. 200 nm thick sections were imaged with a 200kV electron microscope (FEI TF20). Images were collected at a set magnification of 9.6k on a 4kx4k CCD camera set to 2x binning, giving an effective pixel size of 1.76 nm. Dual-axis tilt series (1º steps, ±70º per axis) were acquired using SerialEM. Protomo software was used for aligning projection images and reconstructing tomograms. Visualization/segmentation of objects of interest was performed in Amira. <h3>Results</h3> In addition to classic ID structures, we observed (a) close proximity between gap junctions and mitochondria of opposing cells; (b) a complex network of tubular structures running perpendicular to the long cell axis; these structures showed a hollow interior, with an estimated inner diameter of ~40 nm and were often adjacent to gap junctions or desmosomes; (c) triads formed by lateral edges of gap junctions and desmosomes, with a rough budding vesicle separating the two structures; (d) budding vesicles of approximately 50 nm interrupting the continuity of one side of the gap junction plaque; (e) vesicular bodies of approx. 65 nm in diameter in the intercellular space, and in proximity to gap junction-containing regions. <h3>Conclusions</h3> We describe the nanometric landscape that surrounds gap junctions. We speculate that the connexome includes a physical association with molecules of the mitochondria, desmosome and microtubular network, and propose that microsomes may pass from one cell to another at the ID. Functional characterization of these structures may lead to novel clues as to the mechanisms of inherited or acquired arrhythmias that involve disruption of the ID.