Best Basic Science abstract329Superresolution analysis identifies proliferative changes of excitable membranes (T-tubules) during cardiac remodeling leading to orphaning of Ca2+ release sites and prolonged action potentials

# 329 Superresolution analysis identifies proliferative changes of excitable membranes (T-tubules) during cardiac remodeling leading to orphaning of Ca2+ release sites and prolonged action potentials {#article-title-2} Purpose: Membrane invaginations called transverse tubules (TTs) are essential to couple cellular depolarization to thousands of intracellular Ca2+ release units (CRUs) in ventricular myocytes. While confocal studies suggested loss of TTs as a leading mechanism in heart failure (HF), membrane invaginations have nanometric dimensions which are not resolved by conventional light microscopy. We employed STED superresolution nanoscopy and quantitative analysis strategies which directly address TT changes in different HF models to characterize remodeling mechanisms within clinically relevant time scales over weeks. Methods: Novel superresolution microscopy strategies for TTs in living myocytes and multimodal confocal imaging of TTs and Ca2+ release were used. Thereby, we characterized structural and functional changes as well as different stages of TT remodeling. Both post-MI and post-TAC murine models of HF were investigated early (1 week), intermediate (4 weeks) and late (8 weeks) after intervention versus sham. Computational modeling of 20,000 intracellular CRUs was used to analyze heterogeneous cellular changes to predict the macroscopic outcome for the cell-wide Ca2+ transient and the action potential (AP). Results: STED showed that the mean area of individual TT cross-sections increased significantly starting early, and progressing through 4 weeks to 8 weeks during HF development. Interestingly, the radius size distribution of individual TTs was significantly right-shifted including a second (abnormal) peak which represent distinct, enlarged membrane structures consistent with heterogeneous membrane changes. The cell-wide TT network showed significant total increases of longitudinal components and branching complexity. The CRU associated protein junctophilin-2 was significantly downregulated, whereas ryanodine receptor (RyR2) clusters were increasingly displaced from striations, suggesting heterogeneous orphaning mechanisms of Ca2+ release sites in HF. Computational modeling of increased heterogeneity showed delayed Ca2+ release early and late in diastole, leading to AP prolongation and significant diastolic Ca2+ leak. Conclusions: We identify proliferative mechanisms of TT remodeling in two independent HF models (post-MI; post-TAC). Our data support local subcellular mechanisms of TT reorganization resulting in increasingly heterogeneous membrane structures and defective CRUs including orphaning. The combined TT and CRU changes may directly contribute to dyssynchronous Ca2+ release and AP prolongation, and further set the propensity for afterdepolarizations.