A FRET assay to quantitate levels of the human β-cardiac myosin interacting heads motif based on its near-atomic resolution structure.

In cardiac muscle, many myosin molecules are in a resting or "OFF" state with their catalytic heads in a folded structure known as the interacting heads motif (IHM). Many mutations in the human β-cardiac myosin gene that cause hypertrophic cardiomyopathy (HCM) are thought to destabilize (decrease the population of) the IHM state. The effects of pathogenic mutations on the IHM structural state are often studied using indirect assays, including a single-ATP turnover assay that detects the super-relaxed (SRX) biochemical state of myosin functionally.

Reassessing the unifying hypothesis for hypercontractility caused by myosin mutations in hypertrophic cardiomyopathy.

Human β-cardiac myosin exists in an ON-state where both myosin heads are accessible for interaction with actin, and an OFF-state where the heads are folded back onto their own coiled-coil tail, interacting with each other via an interacting-heads motif (IHM). Hypertrophic cardiomyopathy (HCM) mutations in β-cardiac myosin cause hypercontractility of the heart. Nine years ago, a unifying hypothesis proposed that hypercontractility caused by myosin HCM-associated mutations is primarily due to an increase in the number of ON-state myosin molecules, rather than altered fundamental alterations of functional myosin parameters such as intrinsic motor force, its velocity of movement along actin, or its ATPase turnover rate, all of which impact power output.

Incomplete-penetrant hypertrophic cardiomyopathy G256E mutation causes hypercontractility and elevated mitochondrial respiration.

Determining the pathogenicity of hypertrophic cardiomyopathy-associated mutations in the β-myosin heavy chain () can be challenging due to its variable penetrance and clinical severity. This study investigates the early pathogenic effects of the incomplete-penetrant G256E mutation on myosin function that may trigger pathogenic adaptations and hypertrophy. We hypothesized that the G256E mutation would alter myosin biomechanical function, leading to changes in cellular functions.

Hypertrophic cardiomyopathy mutations Y115H and E497D disrupt the folded-back state of human beta-cardiac myosin allosterically.

At the molecular level, clinical hypercontractility associated with many hypertrophic cardiomyopathy (HCM)-causing mutations in beta-cardiac myosin appears to be driven by their disruptive effect on the energy-conserving, folded-back, super relaxed (SRX) OFF-state of myosin. A pathological increase in force production results from release of heads from this OFF-state, which results in an increase in the number of heads free to interact with actin and produce force. Pathogenic mutations in myosin can conceivably disrupt the OFF-state by (1) directly affecting the intramolecular interfaces stabilizing the folded-back state, or (2) allosterically destabilizing the folded-back state via disruption of diverse conformational states of the myosin motor along its chemomechanical cycle.

Homologous mutations in human β, embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in β-cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman-Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known whether their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human β, embryonic, and perinatal myosin subfragment-1.

Motility Assay to Probe the Calcium Sensitivity of Myosin and Regulated Thin Filaments.

Calcium-dependent activation of the thin filament mediated by the troponin-tropomyosin complex is key in the regulation of actin-myosin based muscle contraction. Perturbations to this system, either physiological (e.g.

Homologous mutations in , embryonic, and perinatal muscle myosins have divergent effects on molecular power generation.

Mutations at a highly conserved homologous residue in three closely related muscle myosins cause three distinct diseases involving muscle defects: R671C in -cardiac myosin causes hypertrophic cardiomyopathy, R672C and R672H in embryonic skeletal myosin cause Freeman Sheldon syndrome, and R674Q in perinatal skeletal myosin causes trismus-pseudocamptodactyly syndrome. It is not known if their effects at the molecular level are similar to one another or correlate with disease phenotype and severity. To this end, we investigated the effects of the homologous mutations on key factors of molecular power production using recombinantly expressed human , embryonic, and perinatal myosin subfragment-1.

Multi-scale models reveal hypertrophic cardiomyopathy MYH7 G256E mutation drives hypercontractility and elevated mitochondrial respiration.

Rationale: Over 200 mutations in the sarcomeric protein β-myosin heavy chain (MYH7) have been linked to hypertrophic cardiomyopathy (HCM). However, different mutations in MYH7 lead to variable penetrance and clinical severity, and alter myosin function to varying degrees, making it difficult to determine genotype-phenotype relationships, especially when caused by rare gene variants such as the G256E mutation.

Objective: This study aims to determine the effects of low penetrant MYH7 G256E mutation on myosin function.

Cryo-EM structure of the folded-back state of human β-cardiac myosin.

To save energy and precisely regulate cardiac contractility, cardiac muscle myosin heads are sequestered in an 'off' state that can be converted to an 'on' state when exertion is increased. The 'off' state is equated with a folded-back structure known as the interacting-heads motif (IHM), which is a regulatory feature of all class-2 muscle and non-muscle myosins. We report here the human β-cardiac myosin IHM structure determined by cryo-electron microscopy to 3.

Cryo-EM structure of the folded-back state of human β-cardiac myosin.

During normal levels of exertion, many cardiac muscle myosin heads are sequestered in an off-state even during systolic contraction to save energy and for precise regulation. They can be converted to an on-state when exertion is increased. Hypercontractility caused by hypertrophic cardiomyopathy (HCM) myosin mutations is often the result of shifting the equilibrium toward more heads in the on-state.

Hypertrophic cardiomyopathy: Mutations to mechanisms to therapies.

Hypertrophic cardiomyopathy (HCM) affects more than 1 in 500 people in the general population with an extensive burden of morbidity in the form of arrhythmia, heart failure, and sudden death. More than 25 years since the discovery of the genetic underpinnings of HCM, the field has unveiled significant insights into the primary effects of these genetic mutations, especially for the myosin heavy chain gene, which is one of the most commonly mutated genes. Our group has studied the molecular effects of HCM mutations on human β-cardiac myosin heavy chain using state-of-the-art biochemical and biophysical tools for the past 10 years, combining insights from clinical genetics and structural analyses of cardiac myosin.

Hypertrophic cardiomyopathy mutations in the pliant and light chain-binding regions of the lever arm of human β-cardiac myosin have divergent effects on myosin function.

Mutations in the lever arm of β-cardiac myosin are a frequent cause of hypertrophic cardiomyopathy, a disease characterized by hypercontractility and eventual hypertrophy of the left ventricle. Here, we studied five such mutations: three in the pliant region of the lever arm (D778V, L781P, and S782N) and two in the light chain-binding region (A797T and F834L). We investigated their effects on both motor function and myosin subfragment 2 (S2) tail-based autoinhibition.

Altered Cardiac Energetics and Mitochondrial Dysfunction in Hypertrophic Cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is a complex disease partly explained by the effects of individual gene variants on sarcomeric protein biomechanics. At the cellular level, HCM mutations most commonly enhance force production, leading to higher energy demands. Despite significant advances in elucidating sarcomeric structure-function relationships, there is still much to be learned about the mechanisms that link altered cardiac energetics to HCM phenotypes.

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.

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.

Single Residue Variation in Skeletal Muscle Myosin Enables Direct and Selective Drug Targeting for Spasticity and Muscle Stiffness.

Muscle spasticity after nervous system injuries and painful low back spasm affect more than 10% of global population. Current medications are of limited efficacy and cause neurological and cardiovascular side effects because they target upstream regulators of muscle contraction. Direct myosin inhibition could provide optimal muscle relaxation; however, targeting skeletal myosin is particularly challenging because of its similarity to the cardiac isoform.

The hypertrophic cardiomyopathy mutations R403Q and R663H increase the number of myosin heads available to interact with actin.

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.

The Myosin Family of Mechanoenzymes: From Mechanisms to Therapeutic Approaches.

Myosins are among the most fascinating enzymes in biology. As extremely allosteric chemomechanical molecular machines, myosins are involved in myriad pivotal cellular functions and are frequently sites of mutations leading to disease phenotypes. Human β-cardiac myosin has proved to be an excellent target for small-molecule therapeutics for heart muscle diseases, and, as we describe here, other myosin family members are likely to be potentially unique targets for treating other diseases as well.

β-Cardiac myosin hypertrophic cardiomyopathy mutations release sequestered heads and increase enzymatic activity.

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.

Deciphering the super relaxed state of human β-cardiac myosin and the mode of action of mavacamten from myosin molecules to muscle fibers.

Mutations in β-cardiac myosin, the predominant motor protein for human heart contraction, can alter power output and cause cardiomyopathy. However, measurements of the intrinsic force, velocity, and ATPase activity of myosin have not provided a consistent mechanism to link mutations to muscle pathology. An alternative model posits that mutations in myosin affect the stability of a sequestered, super relaxed state (SRX) of the protein with very slow ATP hydrolysis and thereby change the number of myosin heads accessible to actin.

Controlling load-dependent kinetics of β-cardiac myosin at the single-molecule level.

Concepts in molecular tension sensing in biology are growing and have their origins in studies of muscle contraction. In the heart muscle, a key parameter of contractility is the detachment rate of myosin from actin, which determines the time that myosin is bound to actin in a force-producing state and, importantly, depends on the load (force) against which myosin works. Here we measure the detachment rate of single molecules of human β-cardiac myosin and its load dependence.

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.

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.