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


OBJECTIVE
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.


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.
Heart Research Center Goettingen, University Medicine Goettingen, Goettingen, Germany.
Eva
E
Wagner
Marcel A
MA
Lauterbach
Tobias
T
Kohl
Volker
V
Westphal
George S B
GS
Williams
Julia H
JH
Steinbrecher
Jan-Hendrik
JH
Streich
Brigitte
B
Korff
Hoang-Trong M
HT
Tuan
Brian
B
Hagen
Stefan
S
Luther
Gerd
G
Hasenfuss
Ulrich
U
Parlitz
M Saleet
MS
Jafri
Stefan W
SW
Hell
W Jonathan
WJ
Lederer
Stephan E
SE
Lehnart
D000200:Action Potentials; D000818:Animals; D051244:Caveolin 3 / Q000378:metabolism; D003198:Computer Simulation; D004195:Disease Models, Animal; D056966:Excitation Contraction Coupling; D005260:Female; D005456:Fluorescent Dyes; D007091:Image Processing, Computer-Assisted; D007425:Intracellular Membranes / Q000378:metabolism / Q000473:pathology*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018613:Microscopy, Confocal / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008870:Microtubules / Q000378:metabolism / Q000473:pathology*; D008955:Models, Cardiovascular; D009203:Myocardial Infarction / Q000378:metabolism / Q000473:pathology* / Q000503:physiopathology; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000473:pathology*; D036103:Nanotechnology; D019837:Ryanodine Receptor Calcium Release Channel / Q000378:metabolism; D013997:Time Factors; D020257:Ventricular Remodeling
D051244:Caveolin 3; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D019837:Ryanodine Receptor Calcium Release Channel; C412507:junctophilin