Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time

Nelson, Shane R.; Li, Amy; Previs, Samantha Beck; Kennedy, Guy G.; Warshaw, David M.

doi:10.1101/2020.05.27.119065

Cited by 10 publications

(14 citation statements)

References 40 publications

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“…The original discovery of the SRX state was based on single ATP turnover experiments in skinned rabbit fast and slow skeletal muscles (10), which was later confirmed by studies in skeletal and cardiac muscle systems from other species (8,(11)(12)(13)(14)(15)(16)(17)(18). The structural basis for this biochemical SRX state is unclear due to a lack of high-resolution atomic-level structure of the myosin.…”

Section: Introductionmentioning

confidence: 99%

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

Gollapudi¹,

Yu²,

Gan³

et al. 2021

Journal of Biological Chemistry

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A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low ionic strength buffers, thereby serving as a reconstituted in-vitro model for muscle thick filament. While these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin sub-fragments, heavy meromyosin (HMM) and myosin subfragment-1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and increased ADP:ATP ratio destabilize the SRX population. Furthermore, wild-type myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small molecule inhibitor, mavacamten, showed that it is not only more effective in increasing myosin SRX population in STFs than in S1 or HMM , but it also increases myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens the mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, disease, and therapeutic conditions.

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Section: Introductionmentioning

confidence: 99%

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

Gollapudi¹,

Yu²,

Gan³

et al. 2021

Journal of Biological Chemistry

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“…Their coordinated action involves most critically the βmys motor powering contraction by ATP free energy transduction into mechanical work and MYBPC3 in a phosphorylation dependent regulatory role. This contractile system subset dynamically adapts contractile force and velocity using myosin step-size modulation 45, 46 and a coordinated βmys/MYBPC3 interaction 11 . Their significance in cardiac muscle contraction is affirmed by the observation that familial hypertrophic cardiomyopathy is most often associated with mutation in one of these two proteins 47 .…”

Section: Discussionmentioning

confidence: 99%

“…It shows 3 directed pathways linking L8 in MYBPC3 with actin binding (L3), energy transduction (SW), and C1 binding (M1) 15 related functional domains in βmys. The peptide linker in the MYBPC3 N-terminus between C1 and C2 (linker 2 or LT) has phosphorylation sites participating in contractile regulation by modulating myosin activity 9-11 and calcium sensitivity 12 . It is reasonable to assume that disrupting C1 binding to βmys could impact the regulatory function on contraction exerted by LT.…”

Section: Discussionmentioning

confidence: 99%

“…MYBPC3 has a linear array of 8 immunoglobulin-like (Ig-like) and 3 fibronectin-like domains ( Fig 2 ). Peptide linker 2 (LT), in the N-terminus linking Ig-like domains C1 and C2, contains phosphorylation sites participating in contractile regulation by modulating myosin activity 9-11 and calcium sensitivity 12 . The MYBPC3 N-terminus associates alternatively with thin filament actin 13, 14 , the myosin motor 15 , and myosin thick filament 13 while the C-terminal domain binds to LM in the thick filament with CX 16 .…”

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confidence: 99%

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Natural variants with 2D correlation genetics identify domains coordinating sarcomere proteins during contraction

Burghardt

2021

Preprint

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Muscle proteins assemble in a sarcomere then by coordinated action produce contraction force to shorten muscle. In the human heart ventriculum, cardiac myosin motor (βmys) repetitively converts ATP free energy into work. Cardiac myosin binding protein C (MYBPC3) in complex with βmys regulates contraction power generation. Their bimolecular complex βmys/MYBPC3 models the contractile system and is used here to study protein coupling. The database for single nucleotide variants (SNVs) in βmys and MYBPC3 surveys human populations worldwide. It consistently records SNV physical characteristics including substituted residue location in the protein functional domain, the side chain substitution, substitution frequency, and human population group, but inconsistently records SNV implicated phenotype and pathology outcomes. A selected consistent subset of the data trains and validates a feed-forward neural network modeling the contraction mechanism. The full database is completed using the model then interpreted probabilistically with a discrete Bayes network to give the SNV probability for a functional domain location given pathogenicity and human population. Co-domains, intra-protein domains coupling βmys and MYBPC3, are identified by their population correlated SNV probability product for given pathogenicity. Divergent genetics in human populations identify co-domain correlates in this method called 2D correlation genetics. Pathogenic and benign SNV data identify three critical regulatory sites, two in MYBPC3 with links to several domains across the βmys motor, and, one in βmys with links to the known MYBPC3 regulatory domain. Critical sites in MYBPC3 are hinges (one known another proposed) sterically enabling regulatory interactions with βmys. The critical site in βmys is the actin binding C-loop, a contact sensor triggering actin-activated myosin ATPase and contraction velocity modulator coordinating also with actin bound tropomyosin. C-loop and MYBPC3 regulatory domain linkage potentially impacts multiple functions across the contractile system. Identification of co-domains in a binary protein complex implies a capacity to estimate spatial proximity constraints for specific dynamic protein interactions in vivo opening another avenue for protein complex structure/function determination.

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“…Thus, they approach the resolution required to map the position of the SRX along the thick-filament backbone. Like Cooke and his team (1), Nelson et al (8) identify two populations of ATP-binding events, a shortlived population with a 26-s RX-like dwell time and a long-lived population with a 146-s SRX-like dwell time. Near the tip of the thick filament, 60% of the myosin ATPase sites were RX-like, whereas 40% were SRX-like.…”

mentioning

confidence: 99%

The Myosin SRX Comes into Focus

Muretta

2020

Biophysical Journal

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The super-relaxed state (SRX) of skeletal and cardiac myosin II thick filaments correlates with diverse physiologic processes in striated muscle. These include thermogenesis (1), stretch-activation (2), post-tetanic potentiation (3), the Frank-Starling relationship in the heart (4), cardiomyopathy (5,6), and the mechanism of action of the clinically relevant small molecules mavacamten, omecamtiv, and blebbistatin (5,7). Defined by its inhibited ATP hydrolysis rate, investigators hypothesize the SRX is a mechanism for parking-like a carenergy-consuming myosin motors when they are not being used. Despite correlating with physiologic phenomena, the relevance of the SRX remains questioned in living muscle. Watching the SRX in real time at the singlemolecule level would help bring its role in muscle contraction into sharper focus by providing a clear view of how and where it functions. In this issue of Biophysical Journal, Nelson et al. (8) publish ''Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time,'' an elegant singlemolecule study that uses super-resolution approaches to map the location of the SRX within skeletal muscle myofibrils, revealing key facets of this enigmatic biochemical state.

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Imaging ATP Consumption in Resting Skeletal Muscle: One Molecule at a Time

Cited by 10 publications

References 40 publications

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

Natural variants with 2D correlation genetics identify domains coordinating sarcomere proteins during contraction

The Myosin SRX Comes into Focus

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