Nanomechanical phenotypes in cMyBP-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 mutant domains. 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, non-pathogenic 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.

Broad therapeutic benefit of myosin inhibition in hypertrophic cardiomyopathy

“…Much less is known about primary pathomechanisms triggered by MYBPC3 variants that do not cause protein haploinsufficiency, which is problematic because they are responsible for a substantial fraction of HCM cases 7,23,26 . To the best of our knowledge, the R502W strain is the first murine model for this type of variants.…”

“…The C-terminal domains of the protein are strongly bound to the thick filament through strong electrostatic interactions with different regions of myosin molecules 54,55 whereas the N-terminal domains are more dynamic and capable of regulating contraction by reversible interactions that stabilize the active state of thin filaments and the inactive state of thick filaments 56 . The central C3-C7 domains of cMyBP-C have traditionally been regarded as mere connectors of the N-and C-terminal regions of the protein (Figure 1B); however, this apparently minor functional role of C3-C7 domains does not agree with the fact that many missense HCM variants, including R502W, target these domains 23,26,57 . Indeed, recent reports have found that the central region of cMyBP-C interacts with myosin, as assessed both biochemically 24,40 and structurally 54 .…”

“…Although further work is needed to unravel specific molecular mechanisms of myosin modulation by central domains of cMyBP-C, our findings support the functional relevance of interactions of myosin and C3-C7 cMyBP-C domains, as well as the proposal that alteration of myosin-cMyBP-C interactions can be a molecular pathomechanism in HCM 40 . Enticingly, the vast majority of candidate pathogenic, non-protein-haploinsufficient variants targeting central domains of cMyBP-C (R495W, R495G, R495Q, R502Q, R810H, R820W, R820Q) 23 affect the surface charge of targeted domains in a similar manner to R502W. This is a typical mechanism of disruption of protein-protein interactions 47 , which suggests that alterations of surface electrostatics could be used to pinpoint rare pathogenic variants in MYH7 and MYBPC3 for which strong clinical cosegregation is not available and that would be otherwise classified as of uncertain significance 23 .…”

“…N-terminal His-tagged C3 and C-terminal His-tagged C0C7 domains of human WT and R502W cMyBP-C were produced and purified as described previously with some modifications 11,24 . Briefly, Rosetta (DE3) E.coli carrying expression vectors pQE80L containing C3 WT or R502W 23 or pET 21a containing C0C7 WT 11 or R502W (introduced by site-directed mutagenesis by PCR) were grown to an absorbance at 600 nm of 0.6-1. Next, cells were induced with 1 mM IPTG for 3 h at 37 °C and at 250 rpm.…”

“…We chose cMyBP-C p.R502W for several reasons. First, previous in vitro studies have shown that p.R502W does not result in changes in mRNA processing nor protein stability, two typical cMyBP-C haploinsufficiency drivers 23,24 . In addition, p.R502W, the most common cause of HCM in humans found in up to 2% of cases 25 , targets the central C3 domain of cMyBP-C (Figure 1B), a hotspot of similar, highly prevalent pathogenic MYBPC3 variants 26 .…”