The mechanics of the heart: zooming in on hypertrophic cardiomyopathy and cMyBP-C.

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.Research in our laboratory on HCM pathomechanisms induced by MYBPC3 variants is funded by the Spanish Ministry of Science and Innovation (MCIN/AEI/10.13039/501100011033) through grant PID2020120426GB-I00 and the Severo Ochoa Program for Centers of Excellence in R&D in its 2015 and 2020 calls (ref. SEV-2015-0505 and ref. CEX2020-001041-S); and by consortium Tec4Bio-CM (S2018/NMT-4443) from the Comunidad de Madrid. This last call is 50% co-financed by the European Social Fund (ESF) and the European Regional Development Fund (ERDF) for the programming period 2014-2020. The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCIN and the Pro CNIC Foundation. CS-C is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. We thank Eli ' as Herrero-Gal ' an for critical feedback. We thank Metello Innocenti for editorial feedback. We thank two anonymous reviewers for their expert feedback.N

Correspondence on "Computational prediction of protein subdomain stability in MYBPC3 enables clinical risk stratification in hypertrophic cardiomyopathy and enhances variant interpretation" by Thompson et al.

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

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.J.A.C. acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) through grants BIO2014– 54768-P, BIO2017–83640-P (AEI/FEDER, UE), EIN2019–102966, RYC-2014–16604, and BFU2017–90692­ REDT, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), and the Comunidad de Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). This work was supported by NIH grants RM1 GM33289 and HL117138 to J.A.S.; a Stanford Dean’s Postdoctoral Fellowship to D.P. and N.N.; and a Stanford Maternal and Child Health Research Institute (MCHRI) Postdoctoral Fellowship (1220552–140-DHPEU) to N.N. Financial support to D.D.S. comes from Eusko Jaurlaritza (Basque Government) through the project IT1254–19, and grants RYC-2016–19590 and PGC2018–099321-B-I00 from the MCIN (FEDER). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCIN, and the Pro CNIC Foundation and was a Severo Ochoa Center of Excellence (SEV-2015–0505). We acknowledge funding from ISCIII to the Centro de Investigación Biomédica en Red (CIBERCV), CB16/11/00425. C.S.C. is the recipient of an FPI-SO predoctoral fellowship, BES-2016–076638. M.R.P. was the recipient of a Ph.D. fellowship from the Italian Ministry of Education, Universities and Research (MIUR). C.P.L. was a recipient of a CNIC Master Fellowship. We thank N. Vicente for excellent technical support (through grant PEJ16/MED/TL-1593 from Consejería de Educación, Juventud y Deporte de la Comunidad de Madrid and the European Social Fund). We thank the Spectroscopy and Nuclear Magnetic Resonance Core Unit at CNIO for access to CD instrumentation and discussion about protein binding assays. We thank A. Thompson and S. Day for their insights. We thank all members of the Molecular Mechanics of the Cardiovascular System team for helpful discussions and the contribution of five anonymous reviewers.S

Basal oxidation of conserved cysteines modulates cardiac titin stiffness and dynamics

Titin, as the main protein responsible for the passive stiffness of the sarcomere, plays a key role in diastolic function and is a determinant factor in the etiology of heart disease. Titin stiffness depends on unfolding and folding transitions of immunoglobulin-like (Ig) domains of the I-band, and recent studies have shown that oxidative modifications of cryptic cysteines belonging to these Ig domains modulate their mechanical properties in vitro. However, the relevance of this mode of titin mechanical modulation in vivo remains largely unknown. Here, we describe the high evolutionary conservation of titin mechanical cysteines and show that they are remarkably oxidized in murine cardiac tissue. Mass spectrometry analyses indicate a similar landscape of basal oxidation in murine and human myocardium. Monte Carlo simulations illustrate how disulfides and S-thiolations on these cysteines increase the dynamics of the protein at physiological forces, while enabling load- and isoform-dependent regulation of titin stiffness. Our results demonstrate the role of conserved cysteines in the modulation of titin mechanical properties in vivo and point to potential redox-based pathomechanisms in heart disease.This work was supported by the Ministerio de Ciencia e Innovación grants BIO2014-54768-P, BIO2017-83640-P, RYC-2014-16604 to JAC and PGC2018-097019-B-I00 to JV, the Regional Government of Madrid grants S2018/NMT-4443 and PEJ16/MED/TL-1593 to JAC and the Instituto de Salud Carlos III (Fondo de Investigación Sanitaria grant PRB3 (PT17/0019/0003- ISCIII-SGEFI /ERDF, ProteoRed), and “la Caixa” Banking Foundation (project code HR17-00247) to JV. We acknowledge funding from the European Research Area Network on Cardiovascular Disease through grant MINOTAUR to SS (The Austrian Science Fund – FWF, I3301) and JAC (ISCIII-AC16/00045). The CNIC is supported by ISCIII, the Ministerio de Ciencia e Innovación and the Pro CNIC Foundation, and was a Severo Ochoa Center of Excellence (SEV-2015-0505). IMM was the recipient of a CNIC-ACCIONA Masters Fellowship and holds a fellowship from “La Caixa” Foundation (ID 100010434, fellowship code LCF/BQ/DR20/11790009). CSC is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. We thank Wolfgang A. Linke and Pablo García-Pavía for critical feedback. We are also thankful for the insights of three anonymous reviewers.S

The Bacterial Mucosal Immunotherapy MV130 Protects Against SARS-CoV-2 Infection and Improves COVID-19 Vaccines Immunogenicity

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.CF was supported by AECC Foundation (INVES192DELF) and is currently funded by the Miguel Servet program (ID: CP20/00106) (ISCIII). IH-M receives the support of a fellowship from la Caixa Foundation (ID 100010434, fellowship code: LCF/BQ/IN17/11620074) and from the European Union’s Horizon 2020 research and innovation program under the Marie Skłodowska-Curie grant agreement no. 713673. AJ-C is a postgraduate fellow of the City Council of Madrid at the Residencia de Estudiantes (2020–2021). GD is supported by an European Molecular Biology Organization (EMBO) Long-term fellowship (ALTF 379-2019). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. Project number 892965. OL and JA-C acknowledge Comunidad de Madrid (Tec4Bio-CM, S2018/NMT-4443, FEDER). Work in OL laboratory was funded by CNIO with the support of the projects Y2018/BIO4747 and P2018/NMT4443 from Comunidad de Madrid and co-funded by the European Social Fund and the European Regional Development Fund. The CNIO is supported by the Instituto de Salud Carlos III (ISCIII). Work at CNB and CISA is funded by the Spanish Health Ministry, Instituto de Salud Carlos III (ISCIII), Fondo COVID-19 grant COV20/00151, and Fondo Supera COVID-19 (Crue Universidades-Banco Santander) (to JG-A). Work in the DS laboratory is funded by the CNIC; by the European Research Council (ERC-2016-Consolidator Grant 725091); by Agencia Estatal de Investigación (PID2019-108157RB); by Comunidad de Madrid (B2017/BMD-3733 Immunothercan-CM); by Fondo Solidario Juntos (Banco Santander); by a research agreement with Inmunotek S.L.; and by Fundació La Marató de TV3 (201723). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), the MICINN, and the Pro CNIC Foundation.Peer reviewe

Fenotipos nanomecánicos en miocardiopatía hipertrófica familiar

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, leída el 01-03-2022Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac muscle disease. Anatomically, it is mainly characterized by left ventricular hypertrophy, while, at the functional level, HCM hearts suffer from hypercontractility and diastolic impairment. Most HCM-causing variants target genes encoding components of the sarcomere, the basic contractile unit of cardiomyocytes. A main HCM gene is MYBPC3 encoding cardiac myosin-binding proteinC (cMyBP-C), a protein that acts as a negative regulator of contraction by slowing down the sliding of actomyosin filaments during sarcomere shortening. While many HCM-causing MYBPC3 variants result in the premature truncation of the protein, leading to degradation and reduced total cMyBP-C content (i.e. protein haploinsufficiency), a significant proportion of the mare nontruncating. The molecular mechanisms by which nontruncating MYBPC3 variants lead to HCM development are still obscure. To improve our understanding of HCM etiology, this doctoral thesis has examined molecular pathomechanisms by which nontruncating MYBPC3 variants cause disease. To this aim, we first explored whether the induction of proteinhaplo insufficiency drivers defines the pathogenicity of nontruncating MYBPC3 variants. Acurated database of nontruncating MYBPC3 variants with a defined pathogenicity status was built to explore the prevalence of variant-induced RNA splicing alterations and protein destabilization, two major haploinsufficiency drivers. These molecular traits were specifically detected in around 50% of the HCM-linked variants, meaning that the induction of these haploinsufficiency drivers can be used as supporting functional evidence of pathogenicity in the classification of genetic variants found in HCM patients...La miocardiopatía hipertrófica (HCM en inglés) es la enfermedad hereditaria más común que afecta al músculo cardiaco. Anatómicamente, se caracteriza por la hipertrofia del ventrículo izquierdo, mientras que, a nivel funcional, ocasiona hipercontractilidad y dificultad de relajación diastólica. La mayoría de las variantes genéticas causantes de HCM afectan a genes que codifican componentes del sarcómero, la unidad contráctil básica de los cardiomiocitos. Uno de los principales genes mutados en HCM es MYBPC3, codificante para la isoforma cardiaca de la proteína C de unión a miosina (cMyBP-C), una proteína que actúa como regulador negativo de la contracción frenando el deslizamiento de los filamentos de actomiosina durante el acortamiento de los sarcómeros. Aunque muchas de las variantes en MYBPC3 causantes de HCM resultan en el truncamiento prematuro de la proteína, causando su degradación y la consecuente reducción de su contenido total (dando lugar a haploinsuficiencia), una proporción significativa de variantes no genera truncamientos. Los mecanismos moleculares por los que estas variantes de no truncamiento llevan al desarrollo de HCM son todavía ampliamente desconocidos. Para mejorar nuestro entendimiento acerca de la etiología de HCM, esta tesis doctoral ha examinado los patomecanismos moleculares por los que las variantes de no truncamiento en MYBPC3 causan enfermedad. Para alcanzar este objetivo, primero exploramos la posibilidad de que la inducción de mecanismos generadores de haploinsuficiencia proteica fuese responsable de la patogenicidad de variantes de no truncamiento en MYBPC3. Para ello, se elaboró una base de datos revisada de variantes de no truncamiento en MYBPC3 con un estado patogénico definido para explorar la prevalencia de alteraciones de splicing de RNA y desestabilización proteica como dos mecanismos principales generadores de haploinsuficiencia inducidos por variantes genéticas. Estos efectos moleculares se detectaron específicamente en alrededor del 50% de las variantes causantes de HCM, lo que implica que la inducción de estos mecanismos generadores de haploinsuficiencia puede interpretarse como una evidencia funcional de patogenicidad en la clasificación de variantes genéticas descritas en pacientes de HCM...Fac. de Ciencias QuímicasTRUEunpu

Concurrent atomic force spectroscopy

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.J.A.-C. acknowledges funding from the Ministerio de Ciencia, Innovación y Universidades (MCNU) through grants BIO2014-54768-P, BIO2017-83640-P (AEI/FEDER, UE), and RYC-2014-16604, the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ISCIII, MINOTAUR, AC16/00045), the Comunidad de Madrid (P2018/NMT-4443), and the CNIC-Severo Ochoa intramural grant program (03-2016 IGP). The CNIC is supported by the Instituto de Salud Carlos III (ISCIII), MCNU and the Pro CNIC Foundation, and is a Severo Ochoa Center of Excellence (SEV-2015-0505). C.P.-L. was a recipient of CNIC Master Fellowship. C.S.-C. is the recipient of an FPI predoctoral fellowship BES-2016-076638. We thank Natalia Vicente for excellent technical support (through grant PEJ16/MED/TL-1593 from Comunidad de Madrid).S

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

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

Protein haploinsufficiency drivers identify MYBPC3 variants that cause hypertrophic cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disease. Variants in MYBPC3, the gene encoding cardiac myosin-binding protein C (cMyBP-C), are the leading cause of HCM. However, the pathogenicity status of hundreds of MYBPC3 variants found in patients remains unknown, as a consequence of our incomplete understanding of the pathomechanisms triggered by HCM-causing variants. Here, we examined 44 nontruncating MYBPC3 variants that we classified as HCM-linked or nonpathogenic according to cosegregation and population genetics criteria. We found that around half of the HCM-linked variants showed alterations in RNA splicing or protein stability, both of which can lead to cMyBP-C haploinsufficiency. These protein haploinsufficiency drivers associated with HCM pathogenicity with 100% and 94% specificity, respectively. Furthermore, we uncovered that 11% of nontruncating MYBPC3 variants currently classified as of uncertain significance in ClinVar induced one of these molecular phenotypes. Our strategy, which can be applied to other conditions induced by protein loss of function, supports the idea that cMyBP-C haploinsufficiency is a fundamental pathomechanism in HCM.J. A.-C. acknowledges funding from the Ministerio de Ciencia e Innovación (MCIN) through grants BIO2014-54768-P ; BIO2017-83640-P/MINECO/AEI/FEDER , UE; EIN2019-102966 and RYC-2014-16604 , the European Research Area Network on Cardiovascular Diseases (ERA-CVD/ ISCIII , Spain MINOTAUR, AC16/00045), and the Comunidad de Madrid (consortium Tec4Bio-CM, S2018/NMT-4443, FEDER). The CNIC is supported by the ISCIII, MCIN, and the Pro CNIC Foundation and was a Severo Ochoa Center of Excellence (SEV-2015-0505). We acknowledge funding from ISCIII to the Centro de Investigación Biomédica en Red (CB16/11/00425). L. S. acknowledges funding from MCIN (BFU2015-63571-P). J. S. acknowledges funding from AEI (PID2019-107293GB-I00), Gobierno de Aragón (E45_17R), and ERDF-InterregV-A POCTEFA, Spain (PIREPRED-EFA086/15). C. S.-C. is the recipient of an FPI-SO predoctoral fellowship BES-2016-076638. M. R. P. was the recipient of a PhD fellowship from the Italian Ministry of Education, Universities and Research , Italy. H. G.-C. is the recipient of an FPU16/04232 doctoral contract from MCIN