Harnessing the molecular-scale resolution of DNA-PAINT to study the structural basis of electrical signals of the healthy and arrhythmic hearts (360G-Wellcome-207684_Z_17_Z)

Single molecule localisation microscopy (SMLM) has revolutionised our understanding of signalling microdomains in many biological systems. Among such molecular systems, are arrays of gap junction (GJ) channels paving the fundamental electrical signalling pathways of the heart. Conventional SMLMs are yet, limited by resolution, unable to visualise individual GJ channels within their native tissue environment. One of the newest SMLM techniques, called DNA-PAINT, promises resolution of ~ 5 nm, sufficient to resolve single proteins within tightly packed arrays or clusters and to visually observe their interactions with other molecules. However, by design, this technique is not suitable for imaging complex tissues like the myocardium. This project aims to implement three possible modifications to the DNA-PAINT protocol to enable in situ optical mapping of GJ isoforms at an unprecedented single- channel-level resolution. DNA-PAINT will be further harnessed to quantify the changes in the phosphorylation states of GJ channels within the ventricular cardiac tissue exhibiting pathological electrical disturbances known as arrhythmias. Both the improved DNA-PAINT protocol and the pilot measurements on GJ remodelling will seed a larger investigation pioneering a direct, in situ correlation of the GJ nanostructure and the disturbed electrical patterns within hearts undergoing life-threatening arrhythmias.