Stimulated emission depletion live-cell super-resolution imaging shows proliferative remodeling of T-tubule membrane structures after myocardial infarction.

RATIONALE: Transverse tubules (TTs) couple electric surface signals to remote intracellular Ca(2+) release units (CRUs). Diffraction-limited imaging studies have proposed loss of TT components as disease mechanism in heart failure (HF). OBJECTIVES: Objectives were to develop quantitative super-resolution strategies for live-cell imaging of TT membranes in intact cardiomyocytes and to show that TT structures are progressively remodeled during HF development, causing early CRU dysfunction. METHODS AND RESULTS: Using stimulated emission depletion (STED) microscopy, we characterized individual TTs with nanometric resolution as direct readout of local membrane morphology 4 and 8 weeks after myocardial infarction (4pMI and 8pMI). Both individual and network TT properties were investigated by quantitative image analysis. The mean area of TT cross sections increased progressively from 4pMI to 8pMI. Unexpectedly, intact TT networks showed differential changes. Longitudinal and oblique TTs were significantly increased at 4pMI, whereas transversal components appeared decreased. Expression of TT-associated proteins junctophilin-2 and caveolin-3 was significantly changed, correlating with network component remodeling. Computational modeling of spatial changes in HF through heterogeneous TT reorganization and RyR2 orphaning (5000 of 20 000 CRUs) uncovered a local mechanism of delayed subcellular Ca(2+) release and action potential prolongation. CONCLUSIONS: This study introduces STED nanoscopy for live mapping of TT membrane structures. During early HF development, the local TT morphology and associated proteins were significantly altered, leading to differential network remodeling and Ca(2+) release dyssynchrony. Our data suggest that TT remodeling during HF development involves proliferative membrane changes, early excitation-contraction uncoupling, and network fracturing.

Dynamics of calcium sparks and calcium leak in the heart.

We present what we believe to be a new mathematical model of Ca(2+) leak from the sarcoplasmic reticulum (SR) in the heart. To our knowledge, it is the first to incorporate a realistic number of Ca(2+)-release units, each containing a cluster of stochastically gating Ca(2+) channels (RyRs), whose biophysical properties (e.g., Ca(2+) sensitivity and allosteric interactions) are informed by the latest molecular investigations. This realistic model allows for the detailed characterization of RyR Ca(2+)-release properties, and shows how this balances reuptake by the SR Ca(2+) pump. Simulations reveal that SR Ca(2+) leak consists of brief but frequent single RyR openings (~3000 cell(-1) s(-1)) that are likely to be experimentally undetectable, and are, therefore, "invisible". We also observe that these single RyR openings can recruit additional RyRs to open, due to elevated local (Ca(2+)), and occasionally lead to the generation of Ca(2+) sparks (~130 cell(-1) s(-1)). Furthermore, this physiological formulation of "invisible" leak allows for the removal of the ad hoc, non-RyR mediated Ca(2+) leak terms present in prior models. Finally, our model shows how Ca(2+) sparks can be robustly triggered and terminated under both normal and pathological conditions. Together, these discoveries profoundly influence how we interpret and understand diverse experimental and clinical results from both normal and diseased hearts.