Mutations in the catalytic domain of human β-cardiac myosin that cause early onset hypertrophic cardiomyopathy significantly increase the fundamental parameters that determine ensemble force and velocity

AbstractHypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. In infants it can be particularly severe and it is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two mutations that cause early-onset HCM, D239N and H251N, affect the mechanical output of human β-cardiac myosin at the molecular level. We observe extremely large increases (25% – 95%) in the actin gliding velocity, single molecule intrinsic force, and ATPase activity of the two mutant myosin motors compared to wild type myosin. In contrast to previous studies of HCM-causing mutations in human β-cardiac myosin, these mutations were striking in that they caused changes in biomechanical parameters that were both greater in magnitude and more uniformly consistent with a hyper-contractile phenotype. In addition, S1-S2 binding studies revealed a significant decrease in affinity of the H251N motor for S2, suggesting that this mutation may further increase hyper-contractility by releasing active motors from a sequestered state. This report shows, for the first time, a clear and significant gain in function for all tested molecular biomechanical parameters due to HCM mutations in human β-cardiac myosin.

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Hypertrophic cardiomyopathy mutations at the folded-back sequestered β-cardiac myosin S1-S2 and S1-S1 interfaces release sequestered heads and increase myosin enzymatic activity

10.1101/537159 ◽

2019 ◽

Cited By ~ 4

Author(s):

Arjun S. Adhikari ◽

Darshan V. Trivedi ◽

Saswata S. Sarkar ◽

Dan Song ◽

Kristina B. Kooiker ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Functional Data ◽

Catalytic Domain ◽

Myosin Head ◽

Intramolecular Interactions ◽

Cardiac Myosin ◽

Myosin S1 ◽

Force Velocity ◽

Cardiomyopathy Mutations ◽

Contractility Of The Heart

AbstractHypertrophic cardiomyopathy (HCM) affects 1 in 500 people and leads to hyper-contractility of the heart. Nearly 40 percent of HCM-causing mutations are found in human β-cardiac myosin. Previous studies looking at the effect of HCM mutations on the force, velocity and ATPase activity of the catalytic domain of human β-cardiac myosin have not shown clear trends leading to hypercontractility at the molecular scale. Here we present functional data showing that four separate HCM mutations located at the myosin head-tail (R249Q, H251N) and head-head (D382Y, R719W) interfaces of a folded-back sequestered state referred to as the interacting heads motif lead to a significant increase in the number of heads functionally accessible for interaction with actin. These results provide evidence that HCM mutations can modulate myosin activity by disrupting intramolecular interactions within the proposed sequestered state, thereby leading to hypercontractility at the molecular level.

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Human hypertrophic cardiomyopathy mutation R712L suppresses the working stroke of cardiac myosin and can be rescued by omecamtiv mecarbil

10.1101/2020.10.02.323782 ◽

2020 ◽

Cited By ~ 1

Author(s):

Aaron Snoberger ◽

Bipasha Barua ◽

Jennifer L. Atherton ◽

Henry Shuman ◽

Eva Forgacs ◽

...

Keyword(s):

Heart Failure ◽

Hypertrophic Cardiomyopathy ◽

Atpase Activity ◽

Single Molecule ◽

Cardiac Failure ◽

Flow Chamber ◽

Severe Form ◽

Gliding Motility ◽

Cardiac Myosin ◽

Omecamtiv Mecarbil

AbstractHypertrophic cardiomyopathies (HCMs) are the leading cause of acute cardiac failure in young individuals. Over 300 mutations throughout β-cardiac myosin, including in the motor domain, are associated with HCM. A β-cardiac myosin motor mutation (R712L) leads to a severe form of HCM. Actin-gliding motility of R712L-myosin is inhibited, despite near normal ATPase kinetics. By optical trapping, the working stroke of R712L-myosin was decreased 4-fold, but actin-attachment durations were normal. A prevalent hypothesis that HCM mutants are hypercontractile is thus not universal. R712 is adjacent to the binding site of the heart failure drug omecamtiv mecarbil (OM). OM suppresses the working stroke of normal β-cardiac myosin, but remarkably, OM rescues the R712L-myosin working stroke. Using a flow chamber to interrogate a single molecule during buffer exchange, we found OM rescue to be reversible. Thus, the R712L mutation uncouples lever arm rotation from ATPase activity and this inhibition is rescued by OM.

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Early-Onset Hypertrophic Cardiomyopathy Mutations Significantly Increase the Velocity, Force, and Actin-Activated ATPase Activity of Human β-Cardiac Myosin

Cell Reports ◽

10.1016/j.celrep.2016.11.040 ◽

2016 ◽

Vol 17 (11) ◽

pp. 2857-2864 ◽

Cited By ~ 37

Author(s):

Arjun S. Adhikari ◽

Kristina B. Kooiker ◽

Saswata S. Sarkar ◽

Chao Liu ◽

Daniel Bernstein ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Atpase Activity ◽

Early Onset ◽

Cardiac Myosin ◽

Cardiomyopathy Mutations

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Effects of Hypertrophic Cardiomyopathy Causing R403Q Mutation on Human Beta-Cardiac Myosin Biomechanical Function: Single Molecule to Ensemble Studies

Biophysical Journal ◽

10.1016/j.bpj.2014.11.2432 ◽

2015 ◽

Vol 108 (2) ◽

pp. 445a

Author(s):

Suman Nag ◽

Ruth Sommese ◽

Shirley Sutton ◽

Kathleen Ruppel ◽

James Spudich

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Single Molecule ◽

Cardiac Myosin

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The molecular basis of hypercontractility caused by the hypertrophic cardiomyopathy mutations R403Q and R663H

10.1101/543413 ◽

2019 ◽

Cited By ~ 2

Author(s):

Saswata S. Sarkar ◽

Darshan V. Trivedi ◽

Makenna M. Morck ◽

Arjun S. Adhikari ◽

Shaik N. Pasha ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Protein C ◽

Binding Protein ◽

Cardiac Myosin ◽

Thin Filaments ◽

Fundamental Parameters ◽

Myosin Binding Protein ◽

Myosin Binding Protein C ◽

Force Velocity ◽

Myosin Binding

AbstractHypertrophic cardiomyopathy (HCM) mutations in ß-cardiac myosin and myosin binding protein-C (MyBP-C) cause hypercontractility of the heart. We show that hypercontractility caused by the HCM myosin mutation R663H cannot be explained by changes in the fundamental parameters such as actin-activated ATPase, intrinsic force, velocity of pure actin or regulated thin filaments, or the pCa50 of the velocity of regulated thin filaments. The same conclusion was made earlier for the HCM myosin mutation R403Q (Nag et al. 2015). Using enzymatic assays for the number of functionally-available heads in purified human ß-cardiac myosin preparations, we provide evidence that both R403Q and R663H HCM myosin mutations cause hypercontractility by increasing the number of functionally-accessible myosin heads. We also demonstrate that the myosin mutation R403Q, but not R663H, ablates the binding of myosin with the C0-C7 fragment of myosin binding protein-C.

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Single-Molecule Mechanics of R403Q Cardiac Myosin Isolated From the Mouse Model of Familial Hypertrophic Cardiomyopathy

Circulation Research ◽

10.1161/01.res.86.7.737 ◽

2000 ◽

Vol 86 (7) ◽

pp. 737-744 ◽

Cited By ~ 140

Author(s):

M. J. Tyska ◽

E. Hayes ◽

M. Giewat ◽

C. E. Seidman ◽

J. G. Seidman ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Mouse Model ◽

Single Molecule ◽

Familial Hypertrophic Cardiomyopathy ◽

Cardiac Myosin

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Hypertrophic cardiomyopathy-linked variants of cardiac myosin-binding protein C3 display altered molecular properties and actin interaction

Biochemical Journal ◽

10.1042/bcj20180685 ◽

2018 ◽

Vol 475 (24) ◽

pp. 3933-3948 ◽

Cited By ~ 4

Author(s):

Sahar I. Da'as ◽

Khalid Fakhro ◽

Angelos Thanassoulas ◽

Navaneethakrishnan Krishnamoorthy ◽

Alaaeldin Saleh ◽

...

Keyword(s):

Hypertrophic Cardiomyopathy ◽

Binding Protein ◽

Clinical Severity ◽

Actin Binding ◽

Cardiac Myosin ◽

C2 Domains ◽

Cardiac Phenotype ◽

Myosin Binding Protein ◽

Myosin Binding ◽

The Impact

The most common inherited cardiac disorder, hypertrophic cardiomyopathy (HCM), is characterized by thickening of heart muscle, for which genetic mutations in cardiac myosin-binding protein C3 (c-MYBPC3) gene, is the leading cause. Notably, patients with HCM display a heterogeneous clinical presentation, onset and prognosis. Thus, delineating the molecular mechanisms that explain how disparate c-MYBPC3 variants lead to HCM is essential for correlating the impact of specific genotypes on clinical severity. Herein, five c-MYBPC3 missense variants clinically associated with HCM were investigated; namely V1 (R177H), V2 (A216T), V3 (E258K), V4 (E441K) and double mutation V5 (V3 + V4), all located within the C1 and C2 domains of MyBP-C, a region known to interact with sarcomeric protein, actin. Injection of the variant complementary RNAs in zebrafish embryos was observed to recapitulate phenotypic aspects of HCM in patients. Interestingly, V3- and V5-cRNA injection produced the most severe zebrafish cardiac phenotype, exhibiting increased diastolic/systolic myocardial thickness and significantly reduced heart rate compared with control zebrafish. Molecular analysis of recombinant C0–C2 protein fragments revealed that c-MYBPC3 variants alter the C0–C2 domain secondary structure, thermodynamic stability and importantly, result in a reduced binding affinity to cardiac actin. V5 (double mutant), displayed the greatest protein instability with concomitant loss of actin-binding function. Our study provides specific mechanistic insight into how c-MYBPC3 pathogenic variants alter both functional and structural characteristics of C0–C2 domains leading to impaired actin interaction and reduced contractility, which may provide a basis for elucidating the disease mechanism in HCM patients with c-MYBPC3 mutations.

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