UC San Diego

The sites of cardiac excitation-contraction (E-C) coupling are composed of sarcoplasmic reticulum (SR)-localized calcium release channels, known as ryanodine receptors (RyRs), coupled to voltage-gated L-type calcium channels (LTCCs) on the sarcolemma in junctional membrane micro- domains termed "couplons". Mounting evidence suggests that the dysregulation of calcium fluxes within these domains is critical in the pathogenesis of heart failure. Despite their essential role in the maintenance of normal myocardial excitation and contractility, our quantitative understanding of couplons is greatly limited due to the formidable technical challenge of imaging and exploring the structure-function relationship of the E-C coupling site. In the work presented here, I developed novel two- and three-dimensional approaches for in-resin, correlated super-resolution fluorescent light microscopy (LM) and electron microscopy (EM) to quantify the distribution of key E-C coupling molecules and reveal their association with membranous organelles in mammalian cardiomyocytes. The imaging of resin-embedded sections with stochastic optical reconstruction microscopy (STORM) was immediately followed by ultrastructural mapping using scanning or transmission EM. Three-dimensional EM data were reconstructed with both array and EM tomography. Correlated imaging using STORM and scanning EM across multiple cells revealed that while most RyRs were mapped within couplons, 21.0 ± 4.5% (n=6) of RyRs were non- junctional. LTCCs were found in couplons, and most NCXs were confined to the non-junctional subdomain of the sarcolemma. The exact localizations of junctional and non- junctional RyRs were further elucidated using correlated STORM and EM tomography, confirming that RyR signals colocalized with "feet" structures visible in couplons at the EM level. Interestingly, a significant population of non-junctional RyRs was found at the inter-membrane junctions between the network SR and the outer membrane of mitochondria. This technique was further applied to study the ultrastructural remodeling and associated RyR reorganization in genetically engineered junctophilin 2 knockout mice, a disease model which mimics abnormal E-C coupling observed in heart failure. The approach presented in this dissertation has facilitated the expansion of our understanding of ion-channel organization in the cardiomyocyte E-C coupling pathway and will pave the way for detailed models of the molecular mechanisms that lead to reduced myocardial contractility in heart failure