Kathleen M. Ruppel

Molecular consequences of the R453C hypertrophic cardiomyopathy mutation on human β-cardiac myosin motor function.

Cardiovascular disorders are the leading cause of morbidity and mortality in the developed world, and hypertrophic cardiomyopathy (HCM) is among the most frequently occurring inherited cardiac disorders. HCM is caused by mutations in the genes encoding the fundamental force-generating machinery of the cardiac muscle, including β-cardiac myosin. Here, we present a biomechanical analysis of the HCM-causing mutation, R453C, in the context of human β-cardiac myosin. We found that this mutation causes a ∼30% decrease in the maximum ATPase of the human β-cardiac subfragment 1, the motor domain of myosin, and a similar percent decrease in the in vitro velocity. The major change in the R453C human β-cardiac subfragment 1 is a 50% increase in the intrinsic force of the motor compared with wild type, with no appreciable change in the stroke size, as observed with a dual-beam optical trap. These results predict that the overall force of the ensemble of myosin molecules in the muscle should be higher in the R453C mutant compared with wild type. Loaded in vitro motility assay confirms that the net force in the ensemble is indeed increased. Overall, this study suggests that the R453C mutation should result in a hypercontractile state in the heart muscle.

Molecular genetic tools for study of the cytoskeleton in Dictyostelium

Publisher Summary Dictyostelium discoideum has a number of features that make it an attractive system for cell biological studies. The ability of Dictyostelium cells to perform active ameboid crawling and chemotaxis has made it a popular system for cell motility and signal transduction studies. The lack of a cell wall and ease of cultivation make Dictyostelium an excellent organism for biochemical approaches, allowing large quantities of material to be obtained and lysed without difficulty. This chapter describes molecular genetic tools that are used for transformation, construction of null cell lines, and expression of the cloned myosin gene fragments. Brief coverage is given to other tools and methods common in the field, but the emphasis is on those approaches that are currently being used. Straightforward gene disruption protocols have been developed and used to construct myosin null lines of Dictyostelium . These techniques have also been successfully applied to the study of other genes in Dictyostelium , such as the α-actinin gene.

COLD-SENSITIVE MUTANTS G680V AND G691C OF DICTYOSTELIUM MYOSIN II CONFER DRAMATICALLY DIFFERENT BIOCHEMICAL DEFECTS

Cold-sensitive myosin mutants represent powerful tools for dissecting discrete deficiencies in myosin function. Biochemical characterization of two such mutants, G680V and G691C, has allowed us to identify separate facets of myosin motor function perturbed by each alteration. Compared with wild type, the G680V myosin exhibits a substantially enhanced affinity for several nucleotides, decreased ATPase activity, and overoccupancy or creation of a novel strongly actin-binding state. The properties of the novel strong binding state are consistent with a partial arrest or pausing at the onset of the mechanical stroke. The G691C mutant, on the other hand, exhibits an elevated basal ATPase indicative of premature phosphate release. By releasing phosphate without a requirement for actin binding, the G691C can bypass the part of the cycle involving the mechanical stroke. The two mutants, despite having alterations in glycine residues separated by only 11 residues, have dramatically different consequences on the mechanochemical cycle.

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

SUMMARY 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.

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

Force parameters of human β-cardiac myosin with the hypertrophic cardiomyopathy mutation R403Q show loss of molecular 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 β-cardiac myosin. We present a detailed characterization of the most debated HCM-causing mutation in human β-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 β-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 β-cardiac myosin contractility as a result of the R403Q mutation.

The myosin mesa and the basis of hypercontractility caused by hypertrophic cardiomyopathy mutations

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 myosins own S2 and MyBP-C, that hold myosin in a sequestered state.

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 Ca2+-activated ADP release rates involving the thin filament human β-cardiac myosin complex. Changes in Ca2+ 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 Ca2+ induced conformational changes in the regulatory thin filament proteins. These changes in calcium sensitivity are independent of active, cycling human β-cardiac myosin.

Multidimensional structure-function relationships in human β-cardiac myosin from population-scale genetic variation

Significance Genetic variants in human β-cardiac myosin, which causes muscle contraction in the heart, can lead to hypertrophic cardiomyopathy (HCM), an inherited heart disease that can cause sudden death. New technologies have generated sequence data for large numbers of patients with HCM and unaffected individuals. In this study, we compare the protein structural locations of genetic variants of patients with HCM and the general population to identify spatial regions of the myosin that have a higher than expected proportion of genetic variants associated with HCM and earlier age at diagnosis. In addition, we develop new methods to interrogate the localization of genetic changes in protein structures. Our study demonstrates the power of combining clinical, genetic, and structural data to gain insight into Mendelian disease. Myosin motors are the fundamental force-generating elements of muscle contraction. Variation in the human β-cardiac myosin heavy chain gene (MYH7) can lead to hypertrophic cardiomyopathy (HCM), a heritable disease characterized by cardiac hypertrophy, heart failure, and sudden cardiac death. How specific myosin variants alter motor function or clinical expression of disease remains incompletely understood. Here, we combine structural models of myosin from multiple stages of its chemomechanical cycle, exome sequencing data from two population cohorts of 60,706 and 42,930 individuals, and genetic and phenotypic data from 2,913 patients with HCM to identify regions of disease enrichment within β-cardiac myosin. We first developed computational models of the human β-cardiac myosin protein before and after the myosin power stroke. Then, using a spatial scan statistic modified to analyze genetic variation in protein 3D space, we found significant enrichment of disease-associated variants in the converter, a kinetic domain that transduces force from the catalytic domain to the lever arm to accomplish the power stroke. Focusing our analysis on surface-exposed residues, we identified a larger region significantly enriched for disease-associated variants that contains both the converter domain and residues on a single flat surface on the myosin head described as the myosin mesa. Notably, patients with HCM with variants in the enriched regions have earlier disease onset than patients who have HCM with variants elsewhere. Our study provides a model for integrating protein structure, large-scale genetic sequencing, and detailed phenotypic data to reveal insight into time-shifted protein structures and genetic disease.

Hypertrophic cardiomyopathy and the myosin mesa: viewing an old disease in a new light

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.

Myosin motor function: structural and mutagenic approaches

Recent advances in three areas of myosin research--structural biology, in vitro motility assays, and mutagenesis--are leading to a new understanding of the molecular mechanism of chemomechanical transduction by this motor protein. Highlights include rational design of mutants using the crystal structure of subfragment 1, combined in vivo and in vitro mutant analyses using Dictyostelium, and the emergence of baculovirus as an in vitro system for expression of mutated mammalian myosins.