Unravelling the ultrastructural details of αT-catenin-deficient cell-cell contacts between heart muscle cells by the use of FIB-SEM.

The intercalated disc is an important structure in cardiomyocytes, as it is essential to maintain correct contraction and proper functioning of the heart. Adhesion and communication between cardiomyocytes are mediated by three main types of intercellular junctions, all residing in the intercalated disc: gap junctions, desmosomes and the areae compositae. Mutations in genes that encode junctional proteins, including αT-catenin (encoded by CTNNA3), have been linked to arrhythmogenic cardiomyopathy and sudden cardiac death. In mice, the loss of αT-catenin in cardiomyocytes leads to impaired heart function, fibrosis, changed expression of desmosomal proteins and increased risk for arrhythmias following ischemia-reperfusion. Currently, it is unclear how the intercalated disc and the intercellular junctions are organised in 3D in the hearts of this αT-catenin knockout (KO) mouse model. In order to scrutinise this, ventricular cardiac tissue of αT-catenin KO mice was used for volume electron microscopy (VEM), making use of Focused Ion Beam Scanning Electron Microscopy (FIB-SEM), allowing a careful 3D reconstruction of the intercalated disc, including gap junctions and desmosomes. Although αT-catenin KO and control mice display a comparable organisation of the sarcomere and the different intercalated disc regions, the folds of the plicae region of the intercalated disc are longer and more narrow in the KO heart, and the pale region between the sarcomere and the intercalated disc is larger. In addition, αT-catenin KO intercalated discs appear to have smaller gap junctions and desmosomes in the plicae region, while gap junctions are larger in the interplicae region of the intercalated disc. Although the reason for this remodelling of the ultrastructure after αT-catenin deletion remains unclear, the excellent resolution of the FIB-SEM technology allows us to reconstruct details that were not reported before. LAY DESCRIPTION: Cardiomyocytes are cells that make up the heart muscle. As the chief cell type of the heart, cardiomyocytes are primarily involved in the contractile function of the heart that enables the pumping of blood around the body. Cardiac muscle cells are connected to each other at their short end by numerous intercellular junctions forming together a structure called the intercalated disc. These intercellular junctions comprise specific protein complexes, which are crucial for both intercellular adhesion and correct contraction of the heart. Imaging by conventional electron microscopy (EM) revealed a heavily folded intercalated disc with apparently random organization of the intercellular junctions. However, this conclusion was based on analysis in two dimensions (2D). 3D information of these structures is needed to unravel their true organization and function. In the present study, we used a more contemporary technique, called volume EM, to image and reconstruct the intercalated discs in 3D. By this approach, EM images are made from a whole block of tissue what differs significantly from classical EM methods that uses only one very thin slice for imaging. Further, we analyzed in comparison to normal mice also a mouse model for cardiomyopathy in which a specific protein of the cardiac intercellular junctions, αT-catenin, is absent. Volume EM revealed that in the hearts of these mice with cardiomyopathy, the finger-like folds of the intercalated disc are longer and thinner compared to control hearts. Also the intercellular junctions on the folded parts of the intercalated disc are smaller and their connection to the striated cytoskeleton seems further away. In conclusion, our volume EM study has expanded our understanding of 3D structures at the intercalated discs and will pave the way for more detailed models of disturbed cell-cell contacts associated with heart failure.