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Hypertrophic cardiomyopathy–causing mutations disrupt a key regulatory off state of myosin in thick filaments., Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin and myosin binding protein-C (MyBP-C) lead to hypercontractility of the heart, an early hallmark of HCM. We show that hypercontractility caused by the HCM-causing mutation R663H cannot be explained by changes in fundamental myosin contractile parameters, much like the HCM-causing mutation R403Q. Using enzymatic assays with purified human β-cardiac myosin, we provide evidence that both 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 MyBP-C. Furthermore, addition of C0-C7 decreases the wild-type myosin basal ATPase single turnover rate, while the mutants do not show a similar reduction. These data suggest that a primary mechanism of action for these mutations is to increase the number of myosin heads functionally available for interaction with actin, which could contribute to hypercontractility.

Summary Hypertrophic cardiomyopathy (HCM) is a heritable cardiovascular disorder that affects 1 in 500 people. A significant percentage of HCM is attributed to mutations in β-cardiac myosin, the motor protein that powers ventricular contraction. This study reports how two early-onset HCM mutations, D239N and H251N, affect the molecular biomechanics of human β-cardiac myosin. We observed significant increases (20%–90%) in actin gliding velocity, intrinsic force, and ATPase activity in comparison to wild-type myosin. Moreover, for H251N, we found significantly lower binding affinity between the S1 and S2 domains of myosin, suggesting that this mutation may further increase hyper-contractility by releasing active motors. Unlike previous HCM mutations studied at the molecular level using human β-cardiac myosin, early-onset HCM mutations lead to significantly larger changes in the fundamental biomechanical parameters and show clear hyper-contractility.

Hypertrophic 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 (IHM) 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, which could lead to hypercontractility at the molecular level., Hypertrophic cardiomyopathy (HCM) leads to hyper-contractility of the heart and is often caused by mutations in human β-cardiac myosin. Here authors show that four separate β-cardiac myosin mutations can modulate myosin activity by disrupting intramolecular interactions.

Hypertrophic 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.

Hypertrophic 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.

The sarcomere of muscle is composed of tens of thousands of myosin motors that self-assemble into thick filaments and interact with surrounding actin-based thin filaments in a dense, near-crystalline hexagonal lattice. Together, these actin-myosin interactions enable large-scale movement and force generation, two primary attributes of muscle. Research on isolated fibres has provided considerable insight into the collective properties of muscle, but how actin-myosin interactions are coordinated in an ensemble remains poorly understood. Here, we show that artificial myosin filaments, engineered using a DNA nanotube scaffold, provide precise control over motor number, type and spacing. Using both dimeric myosin V- and myosin VI-labelled nanotubes, we find that neither myosin density nor spacing has a significant effect on the gliding speed of actin filaments. This observation supports a simple model of myosin ensembles as energy reservoirs that buffer individual stochastic events to bring about smooth, continuous motion. Furthermore, gliding speed increases with cross-bridge compliance, but is limited by Brownian effects. As a first step to reconstituting muscle motility, we demonstrate human β-cardiac myosin-driven gliding of actin filaments on DNA nanotubes.

A working model for β-cardiac myosin in the sequestered state and binding assays reveal interactions between the myosin head and tail that are disrupted by mutations associated with hypertrophic cardiomyopathy. Hypertrophic cardiomyopathy (HCM) is primarily caused by mutations in β-cardiac myosin and myosin-binding protein-C (MyBP-C). Changes in the contractile parameters of myosin measured so far do not explain the clinical hypercontractility caused by such mutations. We propose that hypercontractility is due to an increase in the number of myosin heads (S1) that are accessible for force production. In support of this hypothesis, we demonstrate myosin tail (S2)-dependent functional regulation of actin-activated human β-cardiac myosin ATPase. In addition, we show that both S2 and MyBP-C bind to S1 and that phosphorylation of either S1 or MyBP-C weakens these interactions. Importantly, the S1-S2 interaction is also weakened by four myosin HCM-causing mutations but not by two other mutations. To explain these experimental results, we propose a working structural model involving multiple interactions, including those with myosin's own S2 and MyBP-C, that hold myosin in a sequestered state.

The sarcomere is an exquisitely designed apparatus that is capable of generating force, which in the case of the heart results in the pumping of blood throughout the body. At the molecular level, an ATP-dependent interaction of myosin with actin drives the contraction and force generation of the sarcomere. Over the past six decades, work on muscle has yielded tremendous insights into the workings of the sarcomeric system. We now stand on the cusp where the acquired knowledge of how the sarcomere contracts and how that contraction is regulated can be extended to an understanding of the molecular mechanisms of sarcomeric diseases, such as hypertrophic cardiomyopathy (HCM). In this review we present a picture that combines current knowledge of the myosin mesa, the sequestered state of myosin heads on the thick filament, known as the interacting-heads motif (IHM), their possible interaction with myosin binding protein C (MyBP-C) and how these interactions can be abrogated leading to hyper-contractility, a key clinical manifestation of HCM. We discuss the structural and functional basis of the IHM state of the myosin heads and identify HCM-causing mutations that can directly impact the equilibrium between the 'on state' of the myosin heads (the open state) and the IHM 'off state'. We also hypothesize a role of MyBP-C in helping to maintain myosin heads in the IHM state on the thick filament, allowing release in a graded manner upon adrenergic stimulation. By viewing clinical hyper-contractility as the result of the destabilization of the IHM state, our aim is to view an old disease in a new light.

Hypertrophic cardiomyopathy (HCM) is a genetic disease that affects 1 in 500 people. In infants it is particularly severe and is the leading cause of sudden cardiac death in pediatric populations. A high percentage of HCM is attributed to mutations in beta cardiac myosin, the motor protein responsible for ventricular contraction. This study explores how pediatric-specific HCM mutations in beta cardiac myosin (D239N, H251N, and P710R) alter the biochemical and biomechanical properties of beta cardiac myosin at the molecular and cellular levels. Because these mutations manifest early in life, we hypothesize that they will be more severe than mutations that are not pediatric-specific. We investigated the biomechanical properties of these mutations at the molecular level using a purified S1 domain of human beta cardiac myosin. Specifically, we looked at the effects of these mutations on the ATPase activity and the unloaded velocity (in vitro motility) of beta cardiac S1. Upon comparison with WT beta cardiac S1, we find significant differences between the biomechanical properties of the WT and the pediatric-specific myosin mutations, which are more drastic as compared to mutations that are not pediatric-specific. To fully characterize the system, we are currently performing single molecule optical trap force measurements to determine the intrinsic force of these mutations. Future studies will involve creating iPSC-derived cardiomyocytes to study the mechanics of these mutations at the cellular level. The results from this study will increase the understanding of how genetic mutations that cause HCM lead to the presentation of the disease, and will uncover potential targets for therapeutic design to treat the cause of the disease instead of the symptoms.

Hypertrophic cardiomyopathy is the most frequently occurring inherited cardiovascular disease, with a prevalence of more than one in 500 individuals worldwide. Genetically acquired dilated cardiomyopathy is a related disease that is less prevalent. Both are caused by mutations in the genes encoding the fundamental force-generating protein machinery of the cardiac muscle sarcomere, including human β-cardiac myosin, the motor protein that powers ventricular contraction. Despite numerous studies, most performed with non-human or non-cardiac myosin, there is no clear consensus about the mechanism of action of these mutations on the function of human β-cardiac myosin. We are using a recombinantly expressed human β-cardiac myosin motor domain along with conventional and new methodologies to characterize the forces and velocities of the mutant myosins compared with wild type. Our studies are extending beyond myosin interactions with pure actin filaments to include the interaction of myosin with regulated actin filaments containing tropomyosin and troponin, the roles of regulatory light chain phosphorylation on the functions of the system, and the possible roles of myosin binding protein-C and titin, important regulatory components of both cardiac and skeletal muscles.

Cardiac myosin interacts cyclically with actin in the presence of ATP in the sarcomere to drive heart contraction. Previous fiber studies suggested that the structure and function of myosin in the thick filaments are regulated by its own regulatory light chain (RLC) phosphorylation and through its interaction with myosin binding protein-C (MyBP-C). Interestingly, ∼80% of mutations that cause hypertrophic cardiomyopathy (HCM), a relatively common genetic cardiac disorder characterized by hypercontractility, have been found in the genes encoding human β-cardiac myosin and human cardiac MyBP-C. It has been hypothesized that the heads of the two-headed motor can adopt a sequestered structure where the heads interact asymmetrically with their own tail as Ser-15 residue of RLC remains dephosphorylated, and the N-terminal domains of MyBP-C binds to myosin to stabilize the structure. We designed two different two-headed human β-cardiac myosin motor constructs differing in the length of their S2 tail domains and co-expressed them with both of the human light chains to test the hypothesis that the number of myosin heads available for interaction with actin is regulated by phosphorylation of the RLC and MyBP-C. The shorter construct (2-hep HMM) is devoid of the tail region needed for the interaction of S2 with its S1 heads whereas the longer one (25-hep HMM) has the required length of S2 tail region to allow such interactions. We found that the maximal actin-activated ATPase rate for 2-hep HMM and the single-headed myosin construct S1 are essentially the same, and are only slightly changed by RLC phosphorylation. On the other hand, de-phosphorylated 25-hep HMM has a significantly lower maximal actin-activated ATPase than the 2-hep HMM, consistent with the former having fewer actin-accessible S1 heads than the latter. Additionally, binding experiments of 2-hep and 25-hep HMM with the N-terminal C0-C2 domain of human cardiac MyBP-C showed that the affinity of complex formation is weakened by phosphorylation of either RLC or MyBP-C.

Hypertrophic Cardiomyopathy (HCM) is a common genetic disorder that typically involves left ventricular hypertrophy and cardiac hypercontractility. Mutations in β cardiac myosin heavy chain (β-MyHC) are a major cause of HCM, 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 or more years to develop, the severity of the mutations must be somewhat subtle. We hypothesized that mutations which result in early onset disease may show more severe molecular changes in function compared to later onset mutations. In this work, we performed steady-state and transient kinetic analyses of myosins carrying 1 of 7 missense mutations in the motor domain. Of these 7, 4 have been identified in early onset cardiomyopathy screens. The derived parameters were used to model the ATP driven cross-bridge cycle. 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 occupancy of the force-holding actin.myosin.ADP complex at [Actin] = 3 Kapp along with the closely related Duty Ratio (DR; fraction of myosin in strongly attached force-holding states) and the measured ATPases all change in parallel (in both sign and degree of change) compared to wild type (WT) values. Six of the 7 HCM mutations are clearly distinct from a set of DCM mutations previously characterized.

Hypertrophic (HCM) and dilated (DCM) cardiomyopathies are important causes of heart failure, arrhythmia, and sudden cardiac death. HCM is the most common heritable cardiovascular disorder, affecting 1 in 500 individuals, and is caused primarily by mutations that alter proteins of the cardiac sarcomere. Sarcomeric protein mutations are an increasingly recognized cause of familial DCM as well. HCM is associated with severe thickening of the left ventricular wall, preserved/increased systolic (contractile) and impaired diastolic (relaxation) function of the heart. DCM hearts have dilated left ventricular chambers and suffer from inadequate systolic activity. More than 300 point mutations in beta-cardiac myosin are associated with HCM or DCM. It is believed that HCM and DCM mutations increase and decrease respectively the power output of cardiac myosin which leads to a cascade of downstream signaling that gradually leads to the disease phenotype. To test this hypothesis it is essential to measure the power output of all HCM/DCM mutants in a high throughput manner. To quantitatively measure the load dependent myosin power output, we first improved the loaded in vitro motility assay (LIMA) by replacing load generating molecule alpha-actinin with alpha-catenin, which was generously provided by James Nelson at Stanford University. For accurate-and-fast data analysis, we developed a software called FAST that runs over parallel CPUs. Our initial results indicate that two HCM mutants (R719W, R403Q) have higher power output than the wildtype cardiac myosin. FAST-LIMA will be an essential tool for screening drugs that revert the effects of HCM/DCM mutations on the power output of beta-cardiac myosin.

The generation and coordination of cellular traction forces plays important roles in cell adhesion, cell migration, and extracellular matrix (ECM) remodeling, and hence in the development and repair of biological tissues. Historically, models for how cells generate and sense mechanical force have been derived from observations of cells grown on hard, two-dimensional (2D) surfaces. However, the cytoskeletal geometry of cells embedded in porous, 3D environments is inherently different than those found in 2D. Here we seek to understand the spatio-temporal regulation of cellular traction forces in a 3D environment. We embed primary human fibroblasts in a fluorescently labeled fibrin matrix, and use multicolor, time-lapse confocal microscopy to simultaneously image matrix deformation induced by cellular traction and the dynamics of the acto-myosin cytoskeleton and paxillin-rich cell-matrix adhesions. We observe significant traction forces transduced to the matrix through dynamic actomyosin-rich protrusions. Flux analysis of myosin concentrations in the protrusions reveals recruitment of myosin towards the protrusion tip during extension, and localization of myosin farther from the tip during retraction. Moreover, we observe both retrograde and anterograde movement of F-actin and myosin during cell traction generation, as well as similar bi-directional dynamics for paxillin-rich adhesions. Our data suggests a model of force generation and mechanotransduction different from the canonical continuous lamellipodial retrograde flow implicated in 2D cell migration. Ongoing work examines the role of signal transduction pathways in regulating cellular force generation, with the end goal of understanding how cells couple force generation and mechanotransduction in fully 3D environments.