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.

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COVID-19-specific vaccines are efficient prophylactic weapons against SARS-CoV-2 virus. However, boosting innate responses may represent an innovative way to immediately fight future emerging viral infections or boost vaccines. MV130 is a mucosal immunotherapy, based on a mixture of whole heat-inactivated bacteria, that has shown clinical efficacy against recurrent viral respiratory infections. Herein, we show that the prophylactic intranasal administration of this immunotherapy confers heterologous protection against SARS-CoV-2 infection in susceptible K18-hACE2 mice. Furthermore, in C57BL/6 mice, prophylactic administration of MV130 improves the immunogenicity of two different COVID-19 vaccine formulations targeting the SARS-CoV-2 spike (S) protein, inoculated either intramuscularly or intranasally. Independently of the vaccine candidate and vaccination route used, intranasal prophylaxis with MV130 boosted S-specific responses, including CD8+-T cell activation and the production of S-specific mucosal IgA antibodies. Therefore, the bacterial mucosal immunotherapy MV130 protects against SARS-CoV-2 infection and improves COVID-19 vaccines immunogenicity.

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Mutations in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are a leading cause of HCM. However, it remains challenging to define whether specific gene variants found in patients are pathogenic or not, limiting the reach of cardiovascular genetics in the management of HCM. Here, we have examined cMyBP-C haploinsufficiency drivers in 68 clinically annotated non-truncating variants of MYBPC3. We find that 45% of the pathogenic variants show alterations in RNA splicing or protein stability, which can be linked to pathogenicity with 100% and 94% specificity, respectively. Relevant for variant annotation, we uncover that 9% of non-truncating variants of MYBPC3 currently classified as of uncertain significance induce one of these molecular phenotypes. We propose that alteration of RNA splicing or protein stability caused by MYBPC3 variants provide strong evidence of their pathogenicity, leading to improved clinical management of HCM patients and their families

Hypertrophic cardiomyopathy (HCM), a disease characterized by cardiac muscle hypertrophy and hypercontractility, is the most frequently inherited disorder of the heart. HCM is mainly caused by variants in genes encoding proteins of the sarcomere, the basic contractile unit of cardiomyocytes. The most frequently mutated among them is MYBPC3, which encodes cardiac myosin-binding protein C (cMyBP-C), a key regulator of sarcomere contraction. In this review, we summarize clinical and genetic aspects of HCM and provide updated information on the function of the healthy and HCM sarcomere, as well as on emerging therapeutic options targeting sarcomere mechanical activity. Building on what is known about cMyBP-C activity, we examine different pathogenicity drivers by which MYBPC3 variants can cause disease, focussing on protein haploinsufficiency as a common pathomechanism also in nontruncating variants. Finally, we discuss recent evidence correlating altered cMyBP-C mechanical properties with HCM development.

Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM probes make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM force measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty, even when averaging data from multiple, independent experiments. Compared to traditional AFM, we estimate that concurrent strategies can measure differences in protein mechanical unfolding forces with a 6-fold improvement in accuracy or a 30-fold increase in throughput. Prompted by our results, we demonstrate widely applicable orthogonal fingerprinting strategies for concurrent single-molecule nanomechanical profiling of proteins.

Force-spectroscopy by Atomic Force Microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM probes make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM force measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty, even when averaging data from multiple, independent experiments. Compared to traditional AFM, we estimate that concurrent strategies can measure differences in protein mechanical unfolding forces with a 6-fold improvement in accuracy and a 30-fold increase in throughput. Prompted by our results, we demonstrate widely applicable orthogonal fingerprinting strategies for concurrent single-molecule nanomechanical profiling of proteins.

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.