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

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.

Methods

For the myocardial infarction model, phenotyping, cardiomyocyte isolation, confocal microscopy protocols, and statistics, an expanded Methods section is available in the Online Data Supplement .

STED Microscopy Image Acquisition

A custom STED setup was modified for intracellular TT membrane imaging in isolated cardiomyocytes based on an inverted confocal microscope ( Online Figure IA ). For live-cell STED imaging of TT membranes we tested several dyes, of which di-8-ANEPPS provided (1) bright membrane signals and (2) quiescent cardiomyocytes (see Online Methods ). The STED microscopy laser configuration is summarized in Online Figure IA . Excitation at 490 nm wavelength by a pulsed diode laser (Pico TA 490, Toptica, Munich, Germany) was focused through a 1.4 NA oil objective (PL APO oil 100×, Leica, Wetzlar, Germany). The collected fluorescence was passed through 2 dichroic mirrors, filtered via a 675/60 band pass filter, and recorded by an avalanche photodiode detector (PerkinElmer, Waltham, MA). STED pulses were delivered by a Ti:Sapphire laser (MaiTai, Spectra-Physics, Darmstadt, Germany) operating at 80 MHz emitting at 750 nm. The STED beam was passed through a Vortex phase plate producing a focal doughnut (RPC Photonics, Rochester, NY). Excitation pulses were temporally overlaid with STED pulses by external triggering. Imaging planes were recorded by resonant mirror scanning along the first lateral axis (15.8 kHz, SC-30, Electro-Optical Products Corp, Glendale, NY) and by piezostage scanner along the second lateral axis (P-733–3DD, Physik Instrumente GmbH, Karlsruhe, Germany). If optical aberrations are negligible, the focal plane resolution is estimated to be ≈60 nm, derived from measurements of 20-nm fluorescent beads ( Online Figure IB ).

Quantitative STED Image Analysis of TT Cross-Sections A 2D

Gauss function was fitted to the fluorescence signal distribution of individual transversal TT cross-sections. Full width at half-maximum (FWHM) was determined for longitudinal (X) and transversal (Y) cell directions as indicated in Figure 1B . TT cross-section area was calculated by formula: A=π (0.5·X)·(0.5·Y). (2) For the same TT cross sections, contour lines were computed with a resolution of 120 points using a 50% threshold of the maximal pixel intensity. Using the center of mass, contour lines of a given TT population were superimposed in space (X, Y), presented as 2D probability histogram. Furthermore, the mean circumference of individual TT cross-section contours are reported (Tables). (3) The radius size of individual TT cross sections was determined as the median of 120 contour radii and independent of cell orientation. Radius size distributions were plotted.

Using a nonparametric test

(Mann-Whitney), significant changes in median distribution are reported, and 2-peak Gaussian fitting was used to document deviations (increased heterogeneity) from symmetrical distributions. (4) A separate group of superenlarged highly irregular TT cross sections ( Figure 3C ) was characterized by manual contour analysis and not included in the automated contour data as detailed in the corresponding Results section. For further details, please refer to the Online Methods .

Skeleton and Spatial Orientation Analysis

Two-dimensional skeletons of intact TT networks were extracted from STED images by ImageJ (imagej.nih.gov) and Fiji (pacific.mpi-cbg.de plugins). The following analysis steps were applied: local contrast enhancement (CLAHE), median filtering, automated thresholding, and “skeletonizing” of the binary image. TT skeletons were analyzed by Fiji plug-in algorithm (“directionality”) to quantify spatial orientations of network components. Histogram analysis of orientation probability of TT network components relative to principal cell directions is based on local gradient orientations detected by a 5×5 Sobel filter. Network complexity is analyzed through the amount of triple junctions; STED and confocal imaging data are compared in Online Figure VIII . Computational Modeling of Local Ca 2+ Release Function Local CRU function was investigated through Ca 2+ sparks, Ca 2+ transients, [Ca 2+ ]SR, and AP behavior using the fully stochastic mathematical model of Williams et al, 8 which includes spatial determinants of individual CRU nanodomain organization and a realistic number of 20 000 independent release sites. The whole-cell model examines changes in local subspace [Ca 2+ ] i signaling in HF by implementing previously established changes in ion transport proteins (eg, NCX and SERCA; for details see Online Methods ). Orphaning of RyR2 clusters during HF due to heterogeneous TT and/or RyR2 cluster remodeling was implemented for only 25% of CRUs through a 30-fold increase in subspace volume, whereas 75% of CRUs remained unchanged.

Supplementary Material 01