The D75N and P161S Mutations in the C0-C2 Fragment of cMyBP-C Associated with Hypertrophic Cardiomyopathy Disturb the Thin Filament Activation, Nucleotide Exchange in Myosin, and Actin-Myosin Interaction

Abstract

About half of the mutations that lead to hypertrophic cardiomyopathy (HCM) occur in the MYBPC3 gene. However, the molecular mechanisms of pathogenicity of point mutations in cardiac myosin-binding protein C (cMyBP-C) remain poorly understood. In this study, we examined the effects of the D75N and P161S substitutions in the C0 and C1 domains of cMyBP-C on the structural and functional properties of the C0-C1-m-C2 fragment (C0-C2). Differential scanning calorimetry revealed that these mutations disorder the tertiary structure of the C0-C2 molecule. Functionally, the D75N mutation reduced the maximum sliding velocity of regulated thin filaments in an in vitro motility assay, while the P161S mutation increased it. Both mutations significantly reduced the calcium sensitivity of the actin-myosin interaction and impaired thin filament activation by cross-bridges. D75N and P161S C0-C2 fragments substantially decreased the sliding velocity of the F-actin-tropomyosin filament. ADP dose-dependently reduced filament sliding velocity in the presence of WT and P161S fragments, but the velocity remained unchanged with the D75N fragment. We suppose that the D75N mutation alters nucleotide exchange kinetics by decreasing ADP affinity to the ATPase pocket and slowing the myosin cycle. Our molecular dynamics simulations mean that the D75N mutation affects myosin S1 function. Both mutations impair cardiac contractility by disrupting thin filament activation. The results offer new insights into the HCM pathogenesis caused by missense mutations in N-terminal domains of cMyBP-C, highlighting the distinct effects of D75N and P161S mutations on cardiac contractile function.

Keywords: actin–myosin interaction; cardiac myosin-binding protein C; differential scanning calorimetry; hypertrophic cardiomyopathy mutations; in vitro motility assay; thin filament activation.

Figure 3

Effects of cMyBP-C mutations in the N-terminal part of cMyBP-C on the actin–myosin interaction. (a) Dependence of the sliding velocity of thin filaments over myosin in the in vitro motility assay on the C0-C2 fragment loading concentration at pCa4. (b) Calcium dependence of the sliding velocity of thin filaments over myosin. (c) Effect of cMyBP-C mutations on the relationship between the thin filament sliding velocity and myosin concentration at pCa4. (d) Influence of cMyBP-C mutations on the dependence of the sliding velocity of F-actin–Tpm filaments on myosin concentration. In (a), the experimental data (mean ± SD) are fitted by the logistic function. In (b–d), the data (mean ± SD) are fitted to the Hill equation. The equation parameters are given in Table 1 and Table 2.

Figure 1

Temperature dependences of excess heat capacity (Cp) monitored by DSC for the WT C0-C2 fragment and C0-C2 fragments with D75N and P161S mutations.

Figure 2

Binding of C0-C2 fragments to F-actin. (a) Examples of images of F-actin bound to the flow cell surface at 100 nM, 300 nM, and 500 nM loading concentrations of C0-C2 fragments. (b) The dependence of the mean fluorescence intensity in the microscope field of view on the C0-C2 fragment concentration. The intensity was averaged by 10 fields of view in three experiments. Experimental data (mean ± SD) were fitted using the Hill equation corresponding fits shown as lines.

Figure 4

Effect of saturated Ca²⁺ concentration on the sliding velocity of F-actin over myosin in the presence of 500 nM cMyBP-C fragments. The velocity is presented as the mean ± SD. The symbol * indicates the statistically significant difference between the sliding velocity of F-actin at saturating Ca²⁺ concentration (+Ca²⁺) from those without Ca²⁺ (−Ca²⁺), p < 0.05. Statistical significance was estimated using the Student’s t-test.

Figure 5

(a) Dependence of the sliding velocity of thin filaments on the ATP concentration. The experimental data are fitted to the Hill equation. (b) Dependence of the sliding velocity of thin filaments on the ADP concentration. The experimental data (mean ± SD) for 500 nM D75N C0-C2 fragment are fitted by a linear function; experimental data (mean ± SD) for 0 nM WT C0-C2 fragment, 500 nM WT C0-C2 fragment, and 500 nM P161S C0-C2 fragment were fitted to the Hill equation. The values of the ATP and ADP concentration, at which the velocity was half-maximal, are given in Table 3.

Figure 6

(a) The minimal distance between Glu2, first charged N-terminal residue of C0 domain, and actin surface in MD trajectory. (b,c) Fluctuations of Tpm strands from the actin helix shown as standard deviations of the mean of the azimuthal angles of the residues in two chains of the Tpm strand 1 and 2, respectively, from the actin helix defined by the positions of the K328 residues in the corresponding long pseudo-helical actin strand.