Two Classes of Myosin Inhibitors, Para-nitroblebbistatin and Mavacamten, Stabilize β-Cardiac Myosin in Different Structural and Functional States

Abstract

In addition to a conventional relaxed state, a fraction of myosins in the cardiac muscle exists in a low-energy consuming super-relaxed (SRX) state, which is kept as a reserve pool that may be engaged under sustained increased cardiac demand. The conventional relaxed and the super-relaxed states are widely assumed to correspond to a structure where myosin heads are in an open configuration, free to interact with actin, and a closed configuration, inhibiting binding to actin, respectively. Disruption of the myosin SRX population is an emerging model in different heart diseases, such as hypertrophic cardiomyopathy, which results in excessive muscle contraction, and stabilizing them using myosin inhibitors is budding as an attractive therapeutic strategy. Here we examined the structure-function relationships of two myosin ATPase inhibitors, mavacamten and para-nitroblebbistatin, and found that binding of mavacamten at a site different than para-nitroblebbistatin populates myosin into the SRX state. Para-nitroblebbistatin, binding to a distal pocket to the myosin lever arm near the nucleotide-binding site, does not affect the usual myosin SRX state but instead appears to render myosin into a new, perhaps much more inhibited, 'ultra-relaxed' state. X-ray scattering-based rigid body modeling shows that both mavacamten and para-nitroblebbistatin induce novel conformations in human β-cardiac heavy meromyosin that diverge significantly from the hypothetical open and closed states, and furthermore, mavacamten treatment causes greater compaction than para-nitroblebbistatin. Taken together, we conclude that mavacamten and para-nitroblebbistatin stabilize myosin in different structural states, and such states may give rise to different functional energy-sparing states.

Keywords: Interacting Heads Motif (IHM); Small Angle-X-ray Scattering (SAXS); Super-Relaxed State (SRX); blebbistatin; mavacamten.

Figure 1.. Mavacamten and Para-nitroblebbistatin inhibit Basal, Actin-Activated, and Myofibrillar Myosin ATPase activities.

(A) Basal, (B) Actin-activated, and (C) Myofibrillar ATPase activity. All values are presented in the absolute scale (s⁻¹). BcSTF refers to bovine cardiac synthetic thick filaments, and BcMF refers to bovine cardiac myofibrils. Circles and triangles correspond to mavacamten and para-nitroblebbistatin, respectively. Concentrations of these molecules required to attain half-maximal inhibition (IC₅₀) in the ATPase activity are listed in Table 1. Data are expressed as mean ± SEM (n=8 from two experiments that used single preparation of the same reagent purification).

Figure 2.. Mavacamten, but not Para-nitroblebbistatin, Slows the Release of Mant-Nucleotides from Myosin in a Concentration-dependent Manner.

Representative single turnover fluorescence decay profiles for (A) Mavacamten and (B) Para-nitroblebbistatin. The grey shaded region in panels A & B highlights the differences in fluorescence decay profiles from 200 s onwards. The fluorescence decay profiles were acquired for 40 min but only the data corresponding to the first 20 minutes was shown in panels A & B to highlight important differences. (C) The area under the curve, which qualitatively describes the undissociated mant-nucleotides from myosin in a single turnover, is plotted for the traces shown in panels A & B. Only mavacamten, but not para-nitroblebbistatin, shows a concentration-dependent increase in the undissociated mant nucleotide.

Figure 3.. Mavacamten, but not Para-nitroblebbistatin, Populates Myosin in the SRX State.

(A, B, and C) Show comparison of responses between mavacamten and para-nitroblebbistatin in BcSTF for SRX population (A_slow), DRX rate (k_fast), and SRX rate (k_slow), respectively, while (D, E, and F) show the respective comparisons in BcMF. (G, H, and I) Show a comparison of responses in BcSTF in the presence and absence of 10 μM blebbistatin for A_slow, k_fast, and k_slow, respectively. BcSTF refers to bovine cardiac synthetic thick filaments, and BcMF refers to bovine cardiac myofibrils. The data were presented on the absolute scale (% for amplitude and s⁻¹ for rates). Concentrations of mavacamten required to attain half-maximal change (AC₅₀/IC₅₀) in parameters are listed in Table 1. Data are expressed as mean±SEM (n≥8 from two experiments that used single preparation of the same reagent purification).

Figure 4.. Mavacamten Stabilizes a More Compact Structure of Myosin as Compared to Para-niotroblebbistatin.

(A) Comparison of dimensionless Kratky plots from 25-hep HMM treated with DMSO, para-nitroblebbistatin, and mavacamten. Kratky plots are commonly used for qualitative assessment of flexibility and compactness of biological molecules and are independent of the molecular size. The downward shift of the Kratky plot indicates that 25-hep HMM becomes less flexible, probably more compacted, in the presence of either mavacamten or para-nitroblebbistatin compared to control (DMSO). (B) Comparison of the pair-distance distribution function, P(r), of 25-hep HMM treated with DMSO, para-nitroblebbistatin, and mavacamten. P(r) functions of 25-hep HMM indicate an elongated structure with two globular domains consistent with the classic HMM structure. HMM treated with mavacamten and para-nitroblebbistatin elicited a decrease in the maximum dimension (D_max) by different amounts than the untreated (DMSO) control. D_max is the maximum dimension of the molecule. At least three separate experiments were performed on different batches of samples for each treatment to confirm the reproducibility of the trend. Presented here are single representatives, which also produced the best fits during rigid body modeling shown in Fig. 5.

Figure 5.. Rigid-body Modelling Using the SAXS data Suggests Conformational Heterogeneity and a Higher Propensity for Conformations Closer To the Closed Head Configuration in Mavacamten-treated HMM.

Closed head (A) open head (B) conformation with the 25-hep tail and the GFP used for fitting against SAXS data obtained on untreated (DMSO), para-nitroblebbistatin, and mavacamten treated HMM using CRYSOL [47]. χ² values from CRYSOL indicated. Rigid body models obtained using SASREF [48] performed using open and closed head conformations, respectively, as starting models for HMM treated with (C) DMSO, (D) Para-nitroblebbistatin, and (E) Mavacamten. χ² values from SASREF analysis are presented under the corresponding models.

Figure 6.. Inhibition of Myosin ATPase Caused by Mavacamten, but not Para - nitroblebbistatin, Linearly Correlates with Increase in Myosin SRX Population.

Correlation plot between myosin SRX population and (A) basal myosin ATPase activity and (B) actin-activated ATPase activity for mavacamten (circles) and para-nitroblebbistatin (triangles). Regression analysis showed a strong linear correlation between myosin population in SRX state and ATPase activity for mavacamten (R²≥0.94) but not for para-nitroblebbistatin-treated samples. All data were normalized by the DMSO-treated sample data and presented as mean±SEM (n≥8 from two experiments that used single preparation of the same reagent purification).