Molecular-scale visualization of sarcomere contraction within native cardiomyocytes

Abstract

Sarcomeres, the basic contractile units of striated muscle, produce the forces driving muscular contraction through cross-bridge interactions between actin-containing thin filaments and myosin II-based thick filaments. Until now, direct visualization of the molecular architecture underlying sarcomere contractility has remained elusive. Here, we use in situ cryo-electron tomography to unveil sarcomere contraction in frozen-hydrated neonatal rat cardiomyocytes. We show that the hexagonal lattice of the thick filaments is already established at the neonatal stage, with an excess of thin filaments outside the trigonal positions. Structural assessment of actin polarity by subtomogram averaging reveals that thin filaments in the fully activated state form overlapping arrays of opposite polarity in the center of the sarcomere. Our approach provides direct evidence for thin filament sliding during muscle contraction and may serve as a basis for structural understanding of thin filament activation and actomyosin interactions inside unperturbed cellular environments.

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

“…The “resolution-revolution” of cryogenic electron microscopy (cryo-EM) shook the field of structural biology in recent years, where it became rapidly a dominant technique . Its variant, imaging method cryo-electron tomography (cryo-ET) gives access to the tridimensional reconstruction of cellular interior of plunged-frozen cells and even permits the recognition of macromolecules in situ . − The latest technical developments coupling cryo-EM with light-microscopy and focused ion beam milling have reinforced the capacities of cryo-ET to provide ultrastructure information. ,− Using the latest detectors, electron beam power and lamellar sample sculpting, it is possible to derive tomograms of ∼100–200 nm-thin cell slices at ∼3–4 nm resolution. ,, Cryo-ET studies also reported in-cell structures at nanometer and subnanometer resolutions of amyloid aggregates, − tubulin- or actin-associated proteins, − or membrane protein complexes. − They were obtained using subtomogram averaging from cryo-ET. This approach was initially thought to be less efficient than single-particle cryo-EM to solve structures at high resolution.…”

Structural basis of the super- and hyper-relaxed states of myosin II

“…Incubation with mavacamten may improve its stability ( Anderson et al, 2018 ). How interaction of MyBP-C with myosin enhances the SRX state is another fertile but challenging area of investigation, which may require cryo-electron tomography of thick filaments or intact myofibrils ( Burbaum et al, 2021 ) or single particle cryo-EM studies of MyBP-C–IHM complexes. Experiments suggest that thick filaments in muscle are in equilibrium with a pool of myosin monomer, which may play a role in thick filament assembly/disassembly during development, hypertrophy, and myosin turnover ( Saad et al, 1986 ; Katoh et al, 1998 ; Ojima, 2019 ).…”

Sarcomere maturation: function acquisition, molecular mechanism, and interplay with other organelles

“…The C-terminal of TnI protein constrains the Tm position on the actin filament, and TnI and Tm sterically block myosin from interacting with actin in the absence of Ca 2+ . Contrastingly, Ca 2+ binding leads to a conformational change of TnC, which subsequently releases TnI and Tm from the myosin-binding region of the actin filament, allowing myosin to bind to actin, thereby resulting in sarcomere contraction [17][18][19][20]. A myosin produces the power stroke to slide the actin filament while cross-bridging between actin and thick filaments with ATP hydrolysis [21,22].…”