Abstract 18558: Characterization of Cx43-PKP2 Interaction by Direct Stochastic Optical Reconstruction Microscopy

Abstract

The cardiac intercalated disc is composed of 3 structures involved in the electrical and mechanical coupling of the cardiomyocytes: gap junctions, desmosomes and adherens junctions. Several studies suggest that these structures interact at the intercalated disc. In particular a crosstalk between the desmosomal and gap junction proteins PKP2 and Cx43 has been demonstrated. To characterize the proximity of these two proteins we used a Duolink assay. With this method, we demonstrated that Cx43 and PKP2 reside in the periphery of the adult cardiac intercalated disc at distances <40 nm. To further investigate this interaction we performed direct stochastic optical reconstruction microscopy (dSTORM) experiments. This new approach is a valuable methodology for studying the cellular architecture at the nanoscale. The dSTORM uses standard fluorescent dyes that are controlled by the laser excitation intensity and the imaging buffer conditions can be induced to switch on and off (“blink”). For these experiments, 2.000 images were recorded in a stack, each of these images showing a different subset of the fluorescent dyes active. The analysis of these images (the localization of each blink) gave a reconstructed super-resolution image with a resolution of 10nm. The images obtained in isolated neonatal cardiomyocytes showed that 43.5% of the Cx43 spots/clusters analysed (n=200 of 5 experiments) are an average of 11.38 nm apart from PKP2 (SE= 1.67), 62% of them being in contact (0nm). In conclusion, these two techniques allow us to visualize the interaction and distribution of Cx43 and PKP2 at the nanoscale in the intercalated disc and suggest that a fraction of PKP2 molecules interact directly with Cx43.