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Dissecting β-Cardiac Myosin and Cardiac Myosin-Binding Protein C Interactions using a Nanosurf Assay

Anja M. Touma, Wanjian Tang, David V. Rasicci, Duha Vang, Ashim Rai, Samantha B. Previs, David M. Warshaw, Christopher M. Yengo, Sivaraj Sivaramakrishnan

Preprint posted on March 12, 2022 https://www.biorxiv.org/content/10.1101/2022.03.11.483820v1.full

Nanosurf assay: a new tool to probe how the elusive myosin-binding protein C regulates cardiac contractility

Selected by Neha Nandwani

Background

Cardiac myosin-binding protein C, or cMyBPC, localizes to the C-zone of the cardiac (and skeletal) muscle sarcomeres (Fig A), where it regulates the calcium-activated dynamic interactions between the myosin-containing thick filaments and actin-containing thin filaments that drive muscle contraction (1). Mutations in cMyBPC and β-cardiac myosin are the leading cause of the hypercontractile genetic heart disease hypertrophic cardiomyopathy (HCM), which affects 1 in 200 people and is associated with sudden cardiac death (2). But unlike myosin, the effect of HCM-causing mutations in cMyBPC on the contractile properties of the actomyosin ensembles remains largely uncharacterized, mostly due to the lack of well-suited in vitro assays.

 

cMyBPC has 11 domains (C0-C10); its C-terminal region anchors it to the thick filaments, while the N-terminal region extends away from the thick filaments and spans the space between the thick and thin filaments (Fig B). The N-terminal C0-C3 domains of cMyBPC interact with actin as well as myosin. Decades of research indicates that cMyBPC plays a central role in regulating the strength and dynamics of myocardial contraction. cMyBPC achieves this by contrasting mechanisms (3,4): (i) cMyBPC promotes thin filament sliding at low calcium, likely by interfering with the tropomyosin regulation of thin filament activation, and (ii) cMyBPC inhibits actin filament sliding at high calcium by an undetermined mechanism which limits the velocity of sarcomere shortening. But the molecular details of actin/myosin/cMyBPC interactions are fuzzy because we lack the tools to study these three proteins together in an in vitro setting capable of recapitulating the spatial and stoichiometric constraints observed in the sarcomere.

In this preprint, the authors attempt to provide an inspired solution to this problem: the current study utilizes DNA nanotechnology to design tubular scaffolds displaying recombinant myosin and cMyBPC in a novel modular reconstituted thick filament.

 

Key findings

  1. Previously, the authors described the nanotube assay where DNA origami-derived helical nanotube scaffolds displaying myosin motors via DNA-based protein binding handles were laid on coverslips (Fig. B) and actin filament sliding along nanotubes was measured (5). This is similar in principle, but different in design, from a standard in-vitro motility assay where actin filaments slide over a lawn of myosin motors. However, the frequency of actin-sliding events along nanotubes decorated with human β-cardiac myosin was very low due to its low duty ratio. In the current study, the authors decorate the rest of the coverslip surface with β-cardiac myosin as well (Fig C), which recruits many more actin filaments, ultimately increasing the probability of these filaments encountering the nanotube. This modified assay, termed nanosurf assay, results in a ~100-fold increase in nanotube-associated motile events.
  2. Using distinct DNA-based protein binding handles, the authors generate nanotubes decorated with myosin interdigitated with cMyBPC N-terminal fragments (Fig D). Introduction of the C0-C2 fragment of cMyBPC in the nanotubes reduced the speed at which actin or regulated thin filaments (containing actin, tropomyosin, and troponin) slide over the tubes, similar to what has previously been reported from a standard motility assay. This indicates that the nanosurf assay can be used to gain mechanistic insights into cMyBPC function.
  3. The S2 tail of myosin is known to be the key binding site for cMyBPC. The authors designed nanotubes displaying myosin constructs containing (HMM) or lacking (S1) the proximal S2 tail of myosin and found that the inhibitory effect of cMyBPC on actomyosin nanotube velocity was similar in the two cases. From this, the authors conclude that cMyBPC- actin, but not cMyBPC-myosin, interaction is primarily responsible for the inhibitory effect of cMyBPC on actin sliding.
  4. The authors identify the key role of C1 and C2 domains, linked by the M-domain, in the inhibitory effect of cMyBPC on actin sliding. Unlike previous studies, the authors find that the entire M-domain is important to slow down actin velocity, and reason that the previous results could be artefactual because of non-stoichiometric amounts of cMyBPC fragments used in the assays.
  5. Finally, phosphorylation of cMyBPC at 4 Ser residues within the M-domain accelerates cardiac contractility. Comparing phospho-null and phospho-mimetic N-terminal cMyBPC fragments displayed on nanotubes, the authors show recapitulation of this aspect of cMyBPC regulation of actomyosin inhibition in their nanosurf assays.

 

Why I like this preprint

The field of muscle biophysics will benefit tremendously from an in vitro assay system to monitor myosin, actin and cMyBPC interactions while maintaining their spatial and stoichiometric information. Standard in vitro solution experiments like motility or ATPase assays use very high concentrations of cMyBPC fragments, which may misconstrue the effect cMyBPC has on the actomyosin mechanical activity. On the other hand, intact systems like the isolated native thick filaments from mouse or patient-derived heart tissues are limited by sample availability and are not a practical choice to look at a host of mutations in different proteins. The nanosurf assay not only maintains the spatial relation between the three proteins, but its modularity also makes the introduction of changes in these proteins very simple. The authors have rigorously tested their system against what’s already known and show that this is a robust system. The nanosurf assay can be a powerful tool to investigate the molecular regulation of β-cardiac myosin contractility, and its alteration by disease-causing mutations in cMyBPC and other proteins.

 

Questions for the authors

  1. I am curious to know if the authors have looked at calcium dependence of activation of thin filament sliding in the presence of cMyBPC fragments using the nanosurf assay. I wonder if this system recapitulates the activating effect of cMyBPC at low calcium.
  2. The authors conclude that actin-cMyBPC interaction is the key determinant of the effect of cMyBPC on actin gliding speed in their system. But cMyBPC has been shown to interact with RLC and the motor head in addition to the S2 tail, both of which are present in the S1 construct used to arrive at this conclusion. Have the authors thought of displaying the sS1 construct (which has only the ELC bound) on nanotubes to assess if the loss of RLC affects their conclusion?
  3. Have the authors looked at the potential folding back of the motor heads in their HMM constructs displayed on the nanotubes? A significant fraction of 25-hep HMM exists in the SRX state in vitro under the salt conditions used here (6), which can affect actin-sliding velocity. Further, cMyBPC (and its phosphorylation) has been shown to regulate the equilibrium between the open-closed states of myosin (7). I wonder if this system can be used to probe these regulatory interactions.
  4. I am curious if the authors plan to use the nanosurf assay to characterize small-molecule myosin modulators.

 

References

  1. Harris, P., Lyons, R. G. & Bezold, K. L. In the thick of it: HCM-causing mutations in myosin binding proteins of the thick filament. Circ. Res. 108, 751–764 (2011).
  2. Trivedi, D. V, Adhikari, A. S., Sarkar, S. S., Ruppel, K. M. & Spudich, J. A. Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light. Rev. 10, 27–48 (2018).
  3. Mun, J. Y. et al. Myosin-binding protein C displaces tropomyosin to activate cardiac thin filaments and governs their speed by an independent mechanism. Natl. Acad. Sci. U. S. A. 111, 2170–2175 (2014).
  4. Heling, L. W. H. J., Geeves, M. A. & Kad, N. M. MyBP-C: one protein to govern them all. Muscle Res. Cell Motil. 41, 91–101 (2020).
  5. Hariadi, R. F. et al. Mechanical coordination in motor ensembles revealed using engineered artificial myosin filaments. Nanotechnol. 10, 696–700 (2015).
  6. Anderson, R. L. et al. Deciphering the super relaxed state of human beta-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers. Natl. Acad. Sci. U. S. A. 115, E8143–E8152 (2018).
  7. McNamara, J. W., Singh, R. R. & Sadayappan, S. Cardiac myosin binding protein-C phosphorylation regulates the super-relaxed state of myosin. Natl. Acad. Sci. U. S. A. 116, 11731–11736 (2019).

Tags: actin, hcm, in-vitro reconstitution, myosin, myosin-binding protein c, sarcomere

Posted on: 18th April 2022

doi: https://doi.org/10.1242/prelights.31804

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