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.

Assessment of the Contribution of a Thermodynamic and Mechanical Destabilization of Myosin–Binding Protein C Domain C2 to the Pathomechanism of Hypertrophic Cardiomyopathy–causing Double Mutation MYBPC3Δ25bp/D389V

“…Many of them are frameshift mutations that result in the production of truncated protein, which is degraded in the cell, leading to the phenotype of haploinsufficiency [32][33][34][35][36][37]. In contrast, single amino acid exchanges in MyBPC often alter domain stability or protein-protein interactions, thus exhibiting a poison polypeptide effect [62,[69][70][71].…”

“…This notion is supported by the fact that in relaxed muscle MyBPC extends from the thick filament toward the thin filament, as demonstrated by the use of electron tomography [63]. Using lasertrap assays, the lifetimes of MyBPC's interaction with the thin filament were determined to range from 20 to 300 ms [28] and therefore outlast actomyosin crossbridges (<200 µs) by at least two orders of magnitude [62,64]. It has been proposed that assuming a 10 nm step of the myosin power stroke upon contraction and antiparallel sliding of the filaments, the force applied to MyBPC increases by 250% [62].…”

“…MyBPC is C-terminally anchored to the thick filament via its C8-C10 domains. Additional binding of the N-terminal region of MyBPC to the thin filament establishes a mechanical tether that exerts a viscous load during the cross-bridge cycle [26][27][28]62]. This notion is supported by the fact that in relaxed muscle MyBPC extends from the thick filament toward the thin filament, as demonstrated by the use of electron tomography [63].…”

“…Using lasertrap assays, the lifetimes of MyBPC's interaction with the thin filament were determined to range from 20 to 300 ms [28] and therefore outlast actomyosin crossbridges (<200 µs) by at least two orders of magnitude [62,64]. It has been proposed that assuming a 10 nm step of the myosin power stroke upon contraction and antiparallel sliding of the filaments, the force applied to MyBPC increases by 250% [62]. Under the corresponding mechanical strain, it is possible that subdomains between the anchoring points of the both filaments unfold.…”

“…Using an atomic force microscopy-based approach, this model has been applied to HCM mutations R495W and R502Q in the C3 domain of MyBPC, which shares a similar IgI-like fold with domain C2. The unfolding force as well as the dynamics of unfolding and refolding were examined [62]. Since the C2 domain does not serve as an attachment site on thin filaments, it seems plausible that its function is to act as a brake exerting a set counterforce to cardiac contraction.…”