The calcium signals underpinning the contractile function of cardiomyocytes originate from ryanodine receptors (RyR) located within intracellular signalling sites known as ‘couplons’. Recent super-resolution microscopies imaging have revealed the locations of RyR clusters within the couplons; however fail to fully resolve single RyR channels and partner proteins within these nanodomains due to resolution still limited to ~30 nm. We have achieved greatly enhanced super-resolution imaging based on DNA-PAINT to visualise the nanoscale molecular arrangement of RyR and partner protein junctophilin-2 (JPH2) in peripheral couplons of ventricular cardiomyocytes at a resolution of 5–10 nm. These images reveal an intriguingly non-uniform organisation of RyRs within clusters unlike the crystalline organisation previously predicted based on in vitro experiments. From these measurements, we predict that the communication between RyRs within the couplons is likely to be more complex than previously thought. These images also reveal a very intimate organisation of RyR with the molecular tether JPH2 proteins, thereby providing the first physical evidence that JPH2 remains bound to RyR in the working couplon. The improved resolution was critical to our new observations; DNA-PAINT is a unique tool that can provide a new window into the molecular remodelling that occurs within the couplon in cardiac pathologies.
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