Abstract
Transverse tubules (TTs) are hollow intracellular membrane structures, coupling action potential propagation at the cell surface to intracellular Ca2+ release sites in cardiomyocytes. Since individual TT structures are below the resolution limit of convential light microscopes, the nanoarchitecture in living heart cells is uncertain. Using STED (stimulated emission depletion) nanoscopy and the membrane probe di-8-ANEPPS, we characterized TTs quantitatively deep inside living cardiomyocytes. Using 2D analysis of TT cross-sections we determined diameters and contours indicating regular cylindrical structures. However, after myocardial infarction (post-MI) TT cross-sections appeared dilated. In addition, the continuous TT network changed progressively, resulting in loss of regular tubule orientations and sarcomere misalignement 8 weeks post-MI. In contrast, 4 weeks post-MI a signficant increase of longitudinal network components was evidenced, resembling an immature embryonic phenotype. Analysis of TT associated protein expression confirmed embryonic remodeling 4 weeks post-MI. Furthermore, mathematical modeling of increased spacing between TTs and intracellular Ca2+ release channels revealed delayed subcellular Ca2+ release and action potential prolongation in heart failure. In conclusion, STED nanoscopy of intracellular TT structures in living myocytes provides direct insight about a fundamental mechanisms of excitation-contraction coupling and how TT changes contribute to Ca2+ release dysfunction in heart failure.
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