Correlative super-resolution analysis of cardiac calcium sparks and their molecular origins in health and disease

Rapid release of calcium from internal stores via ryanodine receptors (RyRs) is one of the fastest types of cytoplasmic second messenger signalling in excitable cells. In the heart, rapid summation of the elementary events of calcium release, calcium sparks, determine the contraction of the myocardium. Recent advances in super-resolution microscopy methods have revealed complex clustering patterns of RyRs at single-channel resolution. Here, we have experimentally correlated sub-sarcolemmal spontaneous calcium sparks in rat right ventricular myocytes with the underlying nanoscale sub-sarcolemmal RyR patterns resolved with super-resolution DNA-PAINT microscopy. Local functional groupings and clustering patterns of RyR were sampled by the location and the two-dimensional area of each calcium spark to reveal a steep dependence of the integral of a calcium spark on the sum of the local RyRs. Visualisation of recurring spark sites showed evidence of repeated and triggered saltatory activation of multiple local RyR clusters. In similar myocytes taken from rats with induced right ventricular failure, RyR clusters themselves showed a dissipated morphology and fragmented (smaller) clusters. The failing myocytes featured greater heterogeneity in both the spark properties and the relationship between the integral of the calcium spark and the local ensemble of RyRs. Whilst fragmented (smaller) RyR clusters were rarely observed to directly underlying the larger sparks or the recurring spark sites, local interrogation of the channel-to-channel distances confirmed a clear link between the positions of each calcium spark and the tight non-random clustering of the local RyRs in both healthy and failing ventricles.