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 to-mography 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.

The molecular basis for sarcomere organization in vertebrate skeletal muscle

“…It is well-known from cross-sectional views of myofibrils that thin and thick filaments are hexagonally arranged in the A-band ( Huxley, 1957 ). Interestingly, in immature sarcomeres found within neonatal cardiomyocytes, the hexameric pattern is limited to thick filaments, suggesting that thin filament organization is a later process of sarcomeric maturation in vivo ( Burbaum et al., 2020 ). In orthogonal views of our tomograms, we can clearly discern this hexagonal pattern in the A-band, demonstrating that our preparations are consistent with previous studies ( Figures S1 D–S1F).…”

“…Recent developments in the field have led to a number of structures at resolutions approaching the atomic scale derived from in situ environments and have demonstrated the ability to inform on the molecular and structural details of proteins in their native state and context ( Schur et al., 2016 ; Tegunov et al., 2020 ). Cryo-FIB and cryo-ET were recently employed to investigate neonatal rat cardiomyocytes and provided an initial insight into the organization of muscular filaments inside immature sarcomeres, in which the thick filaments and thin filaments could be observed in a semi-striated arrangement ( Burbaum et al., 2020 ). However, the details of a mature sarcomere from a fully developed vertebrate myofibril, wherein different zones and structures can be distinguished, remain elusive.…”

Single-molecule imaging reveals the concerted release of myosin from regulated thin filaments

“… The very high density of protein within a sarcomere is clear from this image constructed from recent cryo-electron tomography (cryo-ET) measurements of the positions of the sarcomeric components ( Burbaum et al, 2020 ; Wang et al, 2021 ). All components are to scale; thick filaments are shown in yellow, actin filaments in purple/green, and tropomyosin in grey.…”

“…The spatial range of this activation is not clear from the studies here because the fluorescence overlap prevents clear super-resolution of the binding locations. Structural and functional studies have suggested that the active region can be very long ( Desai et al, 2015 ; Vibert et al, 1997 ; Marston, 2003 ); so in the sarcomere with very densely packed heads around the thin filament ( Burbaum et al, 2020 ; Wang et al, 2021 ; Figure 6 ), it is highly possible for multiple heads to bind in close proximity. Therefore, catastrophic collapse could potentially be mediated over a short distance.…”

Cardiomyopathic troponin mutations predominantly occur at its interface with actin and tropomyosin

“…The structure of troponin as bound to the actin filament was derived, in both the presence and absence of Ca 2+ . Although the results may be challenged or revised in time, independent cryo-EM or computational work ( Doran et al, 2020 ; Oda et al, 2020 ; Pavadai et al, 2020a ; Pavadai et al, 2020b ; Burbaum et al, 2020 Preprint ; Wang et al, 2020 Preprint ) is already extending or confirming many aspects, such as the orientation of the troponin core domain. These recent developments are transformative for structural understanding of the primary on-off regulatory switch in cardiac and other striated muscles: reversible Ca 2+ binding to troponin.…”