Quantitative Assessment of Cardiac Intercalated Disk Ultrastructure and Molecular Organization by Indirect Correlative Light and Electron Microscopy

Abstract

The intercalated disk (ID) is a specialized structure that affords electrical (gap junctions) and mechanical (adherens junctions, desmosomes) coupling between cardiomyocytes. Previous research suggests that cardiac sodium channels (NaV1.5) enriched within ID nanodomains may play a key role in cell-to-cell electrical communication. Current models of cardiac conduction do not include the ID or at best, grossly oversimplify it to a uniform flat circle with homogeneous properties. Little experimental data is available to enable realistic in silico representation of the ID and its ion channels. We are using indirect correlative light and electron microscopy (iCLEM) to characterize the ultrastructure (TEM) and molecular organization (STORM) to characterize NaV1.5-rich ID nanodomains under baseline conditions and following acute (60 minutes) structural perturbation using adhesion inhibitor peptides that selectively disrupt different ID nanodomains. Preliminary results indicate that ∼50% of ID-localized NaV1.5 are located within gap junction-adjacent perinexi with another ∼35% being associated with mechanical junctions. Disrupting adhesion within ID nanodomains locally increases intermembrane spacing (electron microscopy), and leads to dynamic dispersal of NaV1.5 clusters (STORM single molecule localization microscopy). In contrast, the inwardly-rectifying potassium channel (Kir2.1) was evenly distributed between GJ- and MJ-associated ID nanodomains and did not undergo redistribution following adhesion inhibitor treatments. Junctional protein distributions were not affected by the peptide inhibitors. Functionally, these effects correlate with conduction slowing and increased arrhythmia vulnerability. Furthermore, the magnitude of functional impacts are likely determined by the amount of sodium channels contained within the nanodomains disrupted. We use these structural data to generate realistic finite-element meshes of ID nanodomain structure and probe their functional roles. This model could reveal previously unanticipated electrical signaling mechanisms at ID nanodomains.