Contractility parameters of human -cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of motor function

Hypertrophic cardiomyopathy (HCM) is the most frequently occurring inherited cardiovascular disease. It is caused by mutations in genes encoding the force-generating machinery of the cardiac sarcomere, including human ?-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human ?-cardiac myosin, R403Q. Despite numerous studies, most performed with nonhuman or noncardiac myosin, there is no consensus about the mechanism of action of this mutation on the function of the enzyme. We use recombinant human ?-cardiac myosin and new methodologies to characterize in vitro contractility parameters of the R403Q myosin compared to wild type. We extend our studies beyond pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin. We find that, with pure actin, the intrinsic force generated by R403Q is ~15% lower than that generated by wild type. The unloaded velocity is, however, ~10% higher for R403Q myosin, resulting in a load-dependent velocity curve that has the characteristics of lower contractility at higher external loads compared to wild type. With regulated actin filaments, there is no increase in the unloaded velocity and the contractility of the R403Q myosin is lower than that of wild type at all loads. Unlike that with pure actin, the actin-activated adenosine triphosphatase activity for R403Q myosin with Ca2+-regulated actin filaments is ~30% lower than that for wild type, predicting a lower unloaded duty ratio of the motor. Overall, the contractility parameters studied fit with a loss of human ?-cardiac myosin contractility as a result of the R403Q mutation

Hypertrophic Cardiomyopathy β-Cardiac Myosin Mutation (P710R) Leads to Hypercontractility by Disrupting Super Relaxed State

Hypertrophic cardiomyopathy (HCM) is the most common inherited form of heart disease, associated with over 1,000 mutations, many in β-cardiac myosin (MYH7). Molecular studies of myosin with different HCM mutations have revealed a diversity of effects on ATPase and load-sensitive rate of detachment from actin. It has been difficult to predict how such diverse molecular effects combine to influence forces at the cellular level and further influence cellular phenotypes. This study focused on the P710R mutation that dramatically decreased in vitro motility velocity and actin-activated ATPase, in contrast to other MYH7 mutations. Optical trap measurements of single myosin molecules revealed that this mutation reduced the step size of the myosin motor and the load sensitivity of the actin detachment rate. Conversely, this mutation destabilized the super relaxed state in longer, two-headed myosin constructs, freeing more heads to generate force. Micropatterned human induced pluripotent derived stem cell (hiPSC)–cardiomyocytes CRISPR-edited with the P710R mutation produced significantly increased force (measured by traction force microscopy) compared with isogenic control cells. The P710R mutation also caused cardiomyocyte hypertrophy and cytoskeletal remodeling as measured by immunostaining and electron microscopy. Cellular hypertrophy was prevented in the P710R cells by inhibition of ERK or Akt. Finally, we used a computational model that integrated the measured molecular changes to predict the measured traction forces. These results confirm a key role for regulation of the super relaxed state in driving hypercontractility in HCM with the P710R mutation and demonstrate the value of a multiscale approach in revealing key mechanisms of disease

A Replication of Failure, Not a Failure to Replicate

Purpose: The increasing role of systematic reviews in knowledge production demands greater rigor in the literature search process. The performance of the Social Work Abstracts (SWA) database has been examined multiple times over the past three decades. The current study is a replication within this line of research. Method: Issue level coverage was examined for the same 33 SWA core journals and the same time period as our 2009 study. Results: The mean percentage of issues missing in the current study was 20%. The mean percentage of issues missing in the current study was significantly greater than the mean percentage of issues missing in the 2009 study. Discussion: The research of other groups, and that of our own, has failed to prompt NASW Press to act. SWA was failing, it is failing and NASW Press has failed to correct those failures

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

Myosin Motor Domains Carrying Mutations Implicated in Early or Late Onset Hypertrophic Cardiomyopathy Have Similar Properties

Hypertrophic cardiomyopathy (HCM) is a common genetic disorder characterized by left ventricular hypertrophy and cardiac hyper-contractility. Mutations in the β cardiac myosin heavy chain gene (β-MyHC) are a major cause of HCM, but the specific mechanistic changes to myosin function that lead to this disease remain incompletely understood. Predicting the severity of any β-MyHC mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, since HCM can take ≥20 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations that result in early onset disease would have more severe changes in function than do later onset mutations. Here, we performed steady-state and transient kinetic analyses of myosins carrying one of seven missense mutations in the motor domain. Of these seven, four were previously identified in early onset cardiomyopathy screens. We used the parameters derived from these analyses to model the ATP driven cross-bridge cycle. Contrary to our hypothesis, the results indicated no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related duty ratio (DR; the fraction of myosin in strongly attached force-holding states) and the measured ATPases all changed in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations

Pediatric and adult-onset HCM mutations in the myosin motor domain have similar properties

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and abnormal cardiac contractility. Mutations in β-MyHC are a major cause of HCM and are typically characterized with cardiac hypercontractility, but the specific mechanistic changes to myosin function that lead to the disease remain incompletely understood. Predicting the severity of any single β-MyHC mutation is hindered by a lack of detailed evaluation at the molecular level. In addition, since the cardiomyopathy can take 20 - 40 years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in childhood cardiomyopathies may show a more severe indication of molecular changes in myosin and be therefore easier to identify. In this work, we performed steady-state and transient kinetics analysis of the myosin carrying one of eight miss sense mutations in the motor domain. Five of these have been identified in childhood cardiomyopathies. The derived parameters were used to model the ATP driven cross bridge. Contrary to our hypothesis, the results show no clear differences between early and late onset HCM mutations. Despite the lack of distinction between early and late onset HCM, the predicted A·M·D occupancy for [A] = 3 Kapp along with the closely related Duty Ratio (DR) and the measured ATPases all change in parallel (in both sign and degree of change) compared to the WT values. Six of the eight HCM mutations are clearly distinct from a set of DCM mutations previously characterized

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity

Dilated cardiomyopathy (DCM) and hypertrophic cardiomyopathy (HCM) can cause arrhythmias, heart failure, and cardiac death. Here, we functionally characterized the motor domains of five DCM-causing mutations in human ?-cardiac myosin. Kinetic analyses of the individual events in the ATPase cycle revealed that each mutation alters different steps in this cycle. For example, different mutations gave enhanced or reduced rate constants of ATP binding, ATP hydrolysis, or ADP release or exhibited altered ATP, ADP, or actin affinity. Local effects dominated, no common pattern accounted for the similar mutant phenotype, and there was no distinct set of changes that distinguished DCM mutations from previously analyzed HCM myosin mutations. That said, using our data to model the complete ATPase contraction cycle revealed additional critical insights. Four of the DCM mutations lowered the duty ratio (the ATPase cycle portion when myosin strongly binds actin) because of reduced occupancy of the force-holding A·M.D complex in the steady-state. Under load, the A·M·D state is predicted to increase owing to a reduced rate constant for ADP release, and this effect was blunted for all five DCM mutations. We observed the opposite effects for two HCM mutations, namely R403Q and R453C. Moreover, the analysis predicted more economical use of ATP by the DCM mutants than by WT and the HCM mutants. Our findings indicate that DCM mutants have a deficit in force generation and force holding capacity due to the reduced occupancy of the force-holding state

Ensemble Force Changes that Result from Human Cardiac Myosin Mutations and a Small-Molecule Effector

Cardiomyopathies due to mutations in human β-cardiac myosin are a significant cause of heart failure, sudden death, and arrhythmia. To understand the underlying molecular basis of changes in the contractile system’s force production due to such mutations and search for potential drugs that restore force generation, an in vitro assay is necessary to evaluate cardiac myosin’s ensemble force using purified proteins. Here, we characterize the ensemble force of human α- and β-cardiac myosin isoforms and those of β-cardiac myosins carrying left ventricular non-compaction (M531R) and dilated cardiomyopathy (S532P) mutations using a utrophin-based loaded in vitro motility assay and new filament-tracking software. Our results show that human α- and β-cardiac myosin, as well as the mutants, show opposite mechanical and enzymatic phenotypes with respect to each other. We also show that omecamtiv mecarbil, a previously discovered cardiac-specific myosin activator, increases β-cardiac myosin force generation

Effects of troponin T cardiomyopathy mutations on the calcium sensitivity of the regulated thin filament and the actomyosin cross-bridge kinetics of human β-cardiac myosin.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca(2+)-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca(2+) sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca(2+) induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin

Effects of troponin T cardiomyopathy mutations on the calcium sensitivity of the regulated thin filament and the actomyosin cross-bridge kinetics of human β-cardiac myosin.

Hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) lead to significant cardiovascular morbidity and mortality worldwide. Mutations in the genes encoding the sarcomere, the force-generating unit in the cardiomyocyte, cause familial forms of both HCM and DCM. This study examines two HCM-causing (I79N, E163K) and two DCM-causing (R141W, R173W) mutations in the troponin T subunit of the troponin complex using human β-cardiac myosin. Unlike earlier reports using various myosin constructs, we found that none of these mutations affect the maximal sliding velocities or maximal Ca(2+)-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca(2+) sensitivity using the human myosin isoform do, however, mimic changes seen previously with non-human myosin isoforms. Transient kinetic measurements show that these mutations alter the kinetics of Ca(2+) induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin