Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy

Abstract

Hypertrophic cardiomyopathy (HCM) is a disease of the myocardium caused by mutations in sarcomeric proteins with mechanical roles, such as the molecular motor myosin. Around half of the HCM-causing genetic variants target contraction modulator cardiac myosin-binding protein C (cMyBP-C), although the underlying pathogenic mechanisms remain unclear since many of these mutations cause no alterations in protein structure and stability. As an alternative pathomechanism, here we have examined whether pathogenic mutations perturb the nanomechanics of cMyBP-C, which would compromise its modulatory mechanical tethers across sliding actomyosin filaments. Using single-molecule atomic force spectroscopy, we have quantified mechanical folding and unfolding transitions in cMyBP-C domains targeted by HCM mutations that do not induce RNA splicing alterations or protein thermodynamic destabilization. Our results show that domains containing mutation R495W are mechanically weaker than wild-type at forces below 40 pN and that R502Q mutant domains fold faster than wild-type. None of these alterations are found in control, nonpathogenic variants, suggesting that nanomechanical phenotypes induced by pathogenic cMyBP-C mutations contribute to HCM development. We propose that mutation-induced nanomechanical alterations may be common in mechanical proteins involved in human pathologies.

Keywords: AFM; cMyBP-C; contraction; hypertrophic cardiomyopathy; protein mechanics; single-molecule.

Figure 1.

Overview of the mechanical role of cMyBP-C in the sarcomere. (a) Comparison of a healthy heart and an HCM counterpart, which shows thicker left ventricular walls and reduced left ventricle volume. Inset: Schematics of the sarcomere, whose contraction relies on actin-based thin filaments that glide over myosin-containing thick filaments thanks to myosin power strokes. cMyBP-C (in yellow) is located in the C-zone, a part of the A-band of the sarcomere. The M-line and the Z-line structures, which arrange filaments supporting sarcomere organization, are also shown. (b) cMyBP-C tethers are subject to mechanical force during a 10 nm myosin power stroke. Interfilament distance is indicated.

Figure 2.

cMyBP-C variants tested in this report. The variants target the C3 central domain of cMyBP-C. Inset: The variants, which induce single nucleotide substitutions in MYBPC3 exon 17, are presented using both cDNA and protein nomenclatures. Variants are colored according to their pathogenicity (red: pathogenic mutations; green: nonpathogenic variants). MYBPC3 exons 16 and 17 code for the C3 domain, and the position of their acceptor (a) and donor (d) splicing sites in the cDNA sequence is indicated. The ribbon diagram presents the immunoglobulin (Ig)-like fold of the C3 domain, in which several β-strands arrange in a Greek key β-sandwich (pdb code 2mq0). The side chains of the residues targeted by the variants are highlighted.

Figure 3.

Characterization of the mechanical stability of WT and mutant C3 domains by single-molecule force spectroscopy by AFM. (a) Left: cMyBP-C tethers experience end-to-end mechanical force during the contraction of actomyosin filaments in systole. The position of the C3 domain within cMyBP-C is indicated. Right: The mechanical properties of a (C3)₈ polyprotein are measured using single-molecule AFM. In these experiments, a single polyprotein is tethered between a cantilever and a moving piezo actuator, and its length is recorded while a linear increase in force is applied. Unfolding events are detected as step increases in the length of 24–25 nm. (b) Cumulative probability of unfolding with force for WT (n = 1033 unfolding events) and R502Q domains (n = 1254 unfolding events) during a 40 pN/s force ramp. (c) Mean unfolding forces in force-ramp experiments, as obtained from Gaussian fits to distributions of unfolding forces (see also Table 1). Error bars correspond to 83% confidence intervals. Bars are colored according to the pathogenic status of the mutation (pathogenic, red; nonpathogenic, green).

Figure 4.

Characterization of r₀ and Δx parameters of WT and mutant C3 domains according to Bell’s model. (a) Distributions of unfolding forces obtained for WT and R502Q domains in a 40 pN/s force ramp. Distributions were fit to Bell’s model (solid lines), and the resulting rate of unfolding at zero force, r₀, and distance to the transition state, Δx, are indicated. (b, c) r₀ and Δx values for WT and mutant C3 domains (see also Supplementary Figure S9 and Table 1). Error bars correspond to 83% confidence intervals. Bars are colored according to the pathogenic status of the mutation (pathogenic, red; nonpathogenic, green). (d) Force dependency of rates of unfolding, r, according to Bell’s model (red: pathogenic, green: nonpathogenic). 83% confidence intervals are indicated as shaded areas.

Figure 5.

Characterization of mechanical folding of WT and mutant C3 domains. (a) Representative trace of mechanical refolding experiments by AFM. A single (C3)₈ polyprotein is subject to an unfolding pulse; then force is quenched to 0 pN, and finally the protein is pulled again to high forces in a probe pulse. Folding fractions are calculated comparing the number of unfolding events in the probe and the unfolding pulses. In the example shown, 5 out of 7 domains refolded during the quench pulse. (b) Folding fractions of WT and R502Q C3 domains at different quench times. Lines are exponential fits to the data. Error bars are SEM estimated by bootstrapping (n ≥ 56 and n ≥ 86 unfolding events for all WT and R502Q data points, respectively). (c) Mechanical folding rates for WT C3 and its mutants, obtained from exponential fits to refolding data (see also Supplementary Figure S11 and Table 1). Error bars are 83% confidence intervals. Bars are colored according to the pathogenic status of the mutation (pathogenic, red; nonpathogenic, green).

Figure 6.

Model of mechanical modulation by cMyBP-C tethers and the influence of domain unfolding. FJC-estimated increase in force generated by a fully folded cMyBP-C tether (black) during a myosin power stroke. If one of the domains of cMyBP-C unfolds, force is reduced (orange). The model considers a radial distribution of domains C3–C7, and that anchoring C2 and C8 domains (not shown for simplicity) are located at the center of the thin and thick filaments, respectively, so that they contribute half their diameter to bridge the 23 nm interfilament space. The increase in cMyBP-C length during a myosin power stroke was estimated using the Pythagorean theorem considering a 10 nm power stroke. In the graph, we also considered that cMyBP-C can dissociate from actin sites at a slower rate than that of myosin power strokes. Mutation R495W increases the rate of mechanical unfolding of C3, whereas R502Q leads to faster C3 folding at low forces.