Publications by authors named "James A Spudich"

94 Publications

Hypertrophic cardiomyopathy β-cardiac myosin mutation (P710R) leads to hypercontractility by disrupting super relaxed state.

Proc Natl Acad Sci U S A 2021 Jun;118(24)

Department of Pediatrics (Cardiology), Stanford University School of Medicine, Palo Alto, CA 94304;

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.
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http://dx.doi.org/10.1073/pnas.2025030118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214707PMC
June 2021

Nanomechanical Phenotypes in Cardiac Myosin-Binding Protein C Mutants That Cause Hypertrophic Cardiomyopathy.

ACS Nano 2021 06 1;15(6):10203-10216. Epub 2021 Jun 1.

Centro Nacional de Investigaciones Cardiovasculares (CNIC), 28029, Madrid, Spain.

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.
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http://dx.doi.org/10.1021/acsnano.1c02242DOI Listing
June 2021

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

Cell 2020 10 8;183(2):335-346.e13. Epub 2020 Oct 8.

MTA-ELTE Motor Pharmacology Research Group, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary; Department of Biochemistry, Eötvös Loránd University, Pázmány Péter sétány 1/c, 1117 Budapest, Hungary and Brunszvik u. 2, 2462 Martonvásár, Hungary. Electronic address:

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. We identified a key residue difference between these myosin isoforms, located in the communication center of the functional regions, which allowed us to design a selective inhibitor, MPH-220. Mutagenic analysis and the atomic structure of MPH-220-bound skeletal muscle myosin confirmed the mechanism of specificity. Targeting skeletal muscle myosin by MPH-220 enabled muscle relaxation, in human and model systems, without cardiovascular side effects and improved spastic gait disorders after brain injury in a disease model. MPH-220 provides a potential nervous-system-independent option to treat spasticity and muscle stiffness.
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http://dx.doi.org/10.1016/j.cell.2020.08.050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7596007PMC
October 2020

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

Sci Adv 2020 04 3;6(14):eaax0069. Epub 2020 Apr 3.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

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.
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http://dx.doi.org/10.1126/sciadv.aax0069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7124958PMC
April 2020

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

Annu Rev Biochem 2020 06 13;89:667-693. Epub 2020 Mar 13.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA; email:

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. The first part of this review focuses on how myosins convert the chemical energy of ATP hydrolysis into mechanical movement, followed by a description of existing therapeutic approaches to target human β-cardiac myosin. The next section focuses on the possibility of targeting nonmuscle members of the human myosin family for several diseases. We end the review by describing the roles of myosin in parasites and the therapeutic potential of targeting them to block parasitic invasion of their hosts.
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http://dx.doi.org/10.1146/annurev-biochem-011520-105234DOI Listing
June 2020

Myosin motor domains carrying mutations implicated in early or late onset hypertrophic cardiomyopathy have similar properties.

J Biol Chem 2019 11 3;294(46):17451-17462. Epub 2019 Oct 3.

BioFrontiers Institute and Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado 80309

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-) 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 mutation is hindered by a lack of detailed examinations at the molecular level. Moreover, because 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 along with the closely related duty ratio (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 with wildtype (WT) values. Six of the seven HCM mutations were clearly distinct from a set of previously characterized DCM mutations.
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http://dx.doi.org/10.1074/jbc.RA119.010563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6873187PMC
November 2019

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

Nat Commun 2019 06 18;10(1):2685. Epub 2019 Jun 18.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.

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.
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http://dx.doi.org/10.1038/s41467-019-10555-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6582153PMC
June 2019

Three perspectives on the molecular basis of hypercontractility caused by hypertrophic cardiomyopathy mutations.

Authors:
James A Spudich

Pflugers Arch 2019 05 15;471(5):701-717. Epub 2019 Feb 15.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.

Several lines of evidence suggest that the primary effect of hypertrophic cardiomyopathy mutations in human β-cardiac myosin is hypercontractility of the heart, which leads to subsequent hypertrophy, fibrosis, and myofilament disarray. Here, I describe three perspectives on the molecular basis of this hypercontractility. The first is that hypercontractility results from changes in the fundamental parameters of the actin-activated β-cardiac myosin chemo-mechanical ATPase cycle. The second considers that hypercontractility results from an increase in the number of functionally accessible heads in the sarcomere for interaction with actin. The final and third perspective is that load dependence of contractility is affected by cardiomyopathy mutations and small-molecule effectors in a manner that changes the power output of cardiac contraction. Experimental approaches associated with each perspective are described along with concepts of therapeutic approaches that could prove valuable in treating hypertrophic cardiomyopathy.
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http://dx.doi.org/10.1007/s00424-019-02259-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6475635PMC
May 2019

SETD3 is an actin histidine methyltransferase that prevents primary dystocia.

Nature 2019 01 10;565(7739):372-376. Epub 2018 Dec 10.

Department of Biology, Stanford University, Stanford, CA, USA.

For more than 50 years, the methylation of mammalian actin at histidine 73 has been known to occur. Despite the pervasiveness of His73 methylation, which we find is conserved in several model animals and plants, its function remains unclear and the enzyme that generates this modification is unknown. Here we identify SET domain protein 3 (SETD3) as the physiological actin His73 methyltransferase. Structural studies reveal that an extensive network of interactions clamps the actin peptide onto the surface of SETD3 to orient His73 correctly within the catalytic pocket and to facilitate methyl transfer. His73 methylation reduces the nucleotide-exchange rate on actin monomers and modestly accelerates the assembly of actin filaments. Mice that lack SETD3 show complete loss of actin His73 methylation in several tissues, and quantitative proteomics analysis shows that actin His73 methylation is the only detectable physiological substrate of SETD3. SETD3-deficient female mice have severely decreased litter sizes owing to primary maternal dystocia that is refractory to ecbolic induction agents. Furthermore, depletion of SETD3 impairs signal-induced contraction in primary human uterine smooth muscle cells. Together, our results identify a mammalian histidine methyltransferase and uncover a pivotal role for SETD3 and actin His73 methylation in the regulation of smooth muscle contractility. Our data also support the broader hypothesis that protein histidine methylation acts as a common regulatory mechanism.
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http://dx.doi.org/10.1038/s41586-018-0821-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511263PMC
January 2019

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

Proc Natl Acad Sci U S A 2018 08 13;115(35):E8143-E8152. Epub 2018 Aug 13.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305;

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. Here we show that purified human β-cardiac myosin exists partly in an SRX and may in part correspond to a folded-back conformation of myosin heads observed in muscle fibers around the thick filament backbone. Mutations that cause hypertrophic cardiomyopathy destabilize this state, while the small molecule mavacamten promotes it. These findings provide a biochemical and structural link between the genetics and physiology of cardiomyopathy with implications for therapeutic strategies.
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http://dx.doi.org/10.1073/pnas.1809540115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6126717PMC
August 2018

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

Nat Struct Mol Biol 2018 06 4;25(6):505-514. Epub 2018 Jun 4.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, USA.

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. We find that both can be modulated by both small-molecule compounds and cardiomyopathy-causing mutations. Furthermore, effects of mutations can be reversed by introducing appropriate compounds. Our results suggest that activating versus inhibitory perturbations of cardiac myosin are discriminated by the aggregate result on duty ratio, average force, and ultimately average power output and suggest that cardiac contractility can be controlled by tuning the load-dependent kinetics of single myosin molecules.
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http://dx.doi.org/10.1038/s41594-018-0069-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092189PMC
June 2018

Dilated cardiomyopathy myosin mutants have reduced force-generating capacity.

J Biol Chem 2018 06 17;293(23):9017-9029. Epub 2018 Apr 17.

the BioFrontiers Institute and Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309,

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 β-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.
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http://dx.doi.org/10.1074/jbc.RA118.001938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5995530PMC
June 2018

Molecular mechanisms and structural features of cardiomyopathy-causing troponin T mutants in the tropomyosin overlap region.

Proc Natl Acad Sci U S A 2017 10 2;114(42):11115-11120. Epub 2017 Oct 2.

Centre for Cardiovascular Biology and Disease, Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India;

Point mutations in genes encoding sarcomeric proteins are the leading cause of inherited primary cardiomyopathies. Among them are mutations in the gene that encodes cardiac troponin T (TnT). These mutations are clustered in the tropomyosin (Tm) binding region of TnT, TNT1 (residues 80-180). To understand the mechanistic changes caused by pathogenic mutations in the TNT1 region, six hypertrophic cardiomyopathy (HCM) and two dilated cardiomyopathy (DCM) mutants were studied by biochemical approaches. Binding assays in the absence and presence of actin revealed changes in the affinity of some, but not all, TnT mutants for Tm relative to WT TnT. HCM mutants were hypersensitive and DCM mutants were hyposensitive to Ca in regulated actomyosin ATPase activities. To gain better insight into the disease mechanism, we modeled the structure of TNT1 and its interactions with Tm. The stability predictions made by the model correlated well with the affinity changes observed in vitro of TnT mutants for Tm. The changes in Ca sensitivity showed a strong correlation with the changes in binding affinity. We suggest the primary reason by which these mutations between residues 92 and 144 cause cardiomyopathy is by changing the affinity of TnT for Tm within the TNT1 region.
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http://dx.doi.org/10.1073/pnas.1710354114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5651771PMC
October 2017

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

Biophys Rev 2018 Feb 17;10(1):27-48. Epub 2017 Jul 17.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA, 94305, USA.

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.
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http://dx.doi.org/10.1007/s12551-017-0274-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5803174PMC
February 2018

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

Nat Struct Mol Biol 2017 Jun 8;24(6):525-533. Epub 2017 May 8.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California, USA.

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.
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http://dx.doi.org/10.1038/nsmb.3408DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737966PMC
June 2017

Biophysical properties of human β-cardiac myosin with converter mutations that cause hypertrophic cardiomyopathy.

Sci Adv 2017 Feb 10;3(2):e1601959. Epub 2017 Feb 10.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

Hypertrophic cardiomyopathy (HCM) affects 1 in 500 individuals and is an important cause of arrhythmias and heart failure. Clinically, HCM is characterized as causing hypercontractility, and therapies are aimed toward controlling the hyperactive physiology. Mutations in the β-cardiac myosin comprise ~40% of genetic mutations associated with HCM, and the converter domain of myosin is a hotspot for HCM-causing mutations; however, the underlying primary effects of these mutations on myosin's biomechanical function remain elusive. We hypothesize that these mutations affect the biomechanical properties of myosin, such as increasing its intrinsic force and/or its duty ratio and therefore the ensemble force of the sarcomere. Using recombinant human β-cardiac myosin, we characterize the molecular effects of three severe HCM-causing converter domain mutations: R719W, R723G, and G741R. Contrary to our hypothesis, the intrinsic forces of R719W and R723G mutant myosins are decreased compared to wild type and unchanged for G741R. Actin and regulated thin filament gliding velocities are ~15% faster for R719W and R723G myosins, whereas there is no change in velocity for G741R. Adenosine triphosphatase activities and the load-dependent velocity change profiles of all three mutant proteins are very similar to those of wild type. These results indicate that the net biomechanical properties of human β-cardiac myosin carrying these converter domain mutations are very similar to those of wild type or are even slightly hypocontractile, leading us to consider an alternative mechanism for the clinically observed hypercontractility. Future work includes how these mutations affect protein interactions within the sarcomere that increase the availability of myosin heads participating in force production.
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http://dx.doi.org/10.1126/sciadv.1601959DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5302870PMC
February 2017

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

Cell Rep 2016 12;17(11):2857-2864

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA; Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; Department of Pediatrics (Cardiology), Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2016.11.040DOI Listing
December 2016

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

Proc Natl Acad Sci U S A 2016 06 31;113(24):6701-6. Epub 2016 May 31.

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305; Stanford Center for Inherited Cardiovascular Disease, Stanford University, Stanford, CA 94305;

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.
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http://dx.doi.org/10.1073/pnas.1606950113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4914177PMC
June 2016

A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.

Science 2016 Feb;351(6273):617-21

Department of Genetics, Harvard Medical School, Boston, MA 02115, USA. Division of Cardiovascular Medicine, Brigham and Women's Hospital, Boston, MA 02115, USA. Howard Hughes Medical Institute, Chevy Chase, MD 20815, USA.

Hypertrophic cardiomyopathy (HCM) is an inherited disease of heart muscle that can be caused by mutations in sarcomere proteins. Clinical diagnosis depends on an abnormal thickening of the heart, but the earliest signs of disease are hyperdynamic contraction and impaired relaxation. Whereas some in vitro studies of power generation by mutant and wild-type sarcomere proteins are consistent with mutant sarcomeres exhibiting enhanced contractile power, others are not. We identified a small molecule, MYK-461, that reduces contractility by decreasing the adenosine triphosphatase activity of the cardiac myosin heavy chain. Here we demonstrate that early, chronic administration of MYK-461 suppresses the development of ventricular hypertrophy, cardiomyocyte disarray, and myocardial fibrosis and attenuates hypertrophic and profibrotic gene expression in mice harboring heterozygous human mutations in the myosin heavy chain. These data indicate that hyperdynamic contraction is essential for HCM pathobiology and that inhibitors of sarcomere contraction may be a valuable therapeutic approach for HCM.
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http://dx.doi.org/10.1126/science.aad3456DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784435PMC
February 2016

Effects of hypertrophic and dilated cardiomyopathy mutations on power output by human β-cardiac myosin.

J Exp Biol 2016 Jan;219(Pt 2):161-7

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA Department of Pediatrics (Cardiology), Stanford University School of Medicine, Stanford, CA 94305, USA

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.
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http://dx.doi.org/10.1242/jeb.125930DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6514469PMC
January 2016

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

Sci Adv 2015 Oct 9;1(9):e1500511. Epub 2015 Oct 9.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

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 Ca(2+)-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.
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http://dx.doi.org/10.1126/sciadv.1500511DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646805PMC
October 2015

Optimized measurements of separations and angles between intra-molecular fluorescent markers.

Nat Commun 2015 Oct 16;6:8621. Epub 2015 Oct 16.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.

We demonstrate a novel, yet simple tool for the study of structure and function of biomolecules by extending two-colour co-localization microscopy to fluorescent molecules with fixed orientations and in intra-molecular proximity. From each colour-separated microscope image in a time-lapse movie and using only simple means, we simultaneously determine both the relative (x,y)-separation of the fluorophores and their individual orientations in space with accuracy and precision. The positions and orientations of two domains of the same molecule are thus time-resolved. Using short double-stranded DNA molecules internally labelled with two fixed fluorophores, we demonstrate the accuracy and precision of our method using the known structure of double-stranded DNA as a benchmark, resolve 10-base-pair differences in fluorophore separations, and determine the unique 3D orientation of each DNA molecule, thereby establishing short, double-labelled DNA molecules as probes of 3D orientation of anything to which one can attach them firmly.
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http://dx.doi.org/10.1038/ncomms9621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4634324PMC
October 2015

Establishing disease causality for a novel gene variant in familial dilated cardiomyopathy using a functional in-vitro assay of regulated thin filaments and human cardiac myosin.

BMC Med Genet 2015 Oct 26;16:97. Epub 2015 Oct 26.

Departments of Medicine (Cardiovascular Medicine), Stanford University School of Medicine, Stanford, CA, USA.

Background: As next generation sequencing for the genetic diagnosis of cardiovascular disorders becomes more widely used, establishing causality for putative disease causing variants becomes increasingly relevant. Diseases of the cardiac sarcomere provide a particular challenge in this regard because of the complexity of assaying the effect of genetic variants in human cardiac contractile proteins.

Results: In this study we identified a novel variant R205Q in the cardiac troponin T gene (TNNT2). Carriers of the variant allele exhibited increased chamber volumes associated with decreased left ventricular ejection fraction. To clarify the causal role of this variant, we generated recombinant variant human protein and examined its calcium kinetics as well as the maximally activated ADP release of human β-cardiac myosin with regulated thin filaments containing the mutant troponin T. We found that the R205Q mutation significantly decreased the calcium sensitivity of the thin filament by altering the effective calcium dissociation kinetics.

Conclusions: The development of moderate throughput post-genomic assays is an essential step in the realization of the potential of next generation sequencing. Although technically challenging, biochemical and functional assays of human cardiac contractile proteins of the thin filament can be achieved and provide an orthogonal source of information to inform the question of causality for individual variants.
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http://dx.doi.org/10.1186/s12881-015-0243-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4620603PMC
October 2015

Harmonic force spectroscopy measures load-dependent kinetics of individual human β-cardiac myosin molecules.

Nat Commun 2015 Aug 4;6:7931. Epub 2015 Aug 4.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305, USA.

Molecular motors are responsible for numerous cellular processes from cargo transport to heart contraction. Their interactions with other cellular components are often transient and exhibit kinetics that depend on load. Here, we measure such interactions using 'harmonic force spectroscopy'. In this method, harmonic oscillation of the sample stage of a laser trap immediately, automatically and randomly applies sinusoidally varying loads to a single motor molecule interacting with a single track along which it moves. The experimental protocol and the data analysis are simple, fast and efficient. The protocol accumulates statistics fast enough to deliver single-molecule results from single-molecule experiments. We demonstrate the method's performance by measuring the force-dependent kinetics of individual human β-cardiac myosin molecules interacting with an actin filament at physiological ATP concentration. We show that a molecule's ADP release rate depends exponentially on the applied load, in qualitative agreement with cardiac muscle, which contracts with a velocity inversely proportional to external load.
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http://dx.doi.org/10.1038/ncomms8931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532873PMC
August 2015

Ensemble force changes that result from human cardiac myosin mutations and a small-molecule effector.

Cell Rep 2015 May 30;11(6):910-920. Epub 2015 Apr 30.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA. Electronic address:

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.
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http://dx.doi.org/10.1016/j.celrep.2015.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4431957PMC
May 2015

The myosin mesa and a possible unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy.

Authors:
James A Spudich

Biochem Soc Trans 2015 Feb;43(1):64-72

*Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, U.S.A.

No matter how many times one explores the structure of the myosin molecule, there is always something new to discover. Here, I describe the myosin mesa, a structural feature of the motor domain that has the characteristics of a binding domain for another protein, possibly myosin-binding protein C (MyBP-C). Interestingly, many well-known hypertrophic cardiomyopathy (HCM) mutations lie along this surface and may affect the putative interactions proposed here. A potential unifying hypothesis for the molecular basis of human hypertrophic cardiomyopathy is discussed here. It involves increased power output of the cardiac muscle as a result of HCM mutations causing the release of inhibition by myosin binding protein C.
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http://dx.doi.org/10.1042/BST20140324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4349527PMC
February 2015

Mechanistic heterogeneity in contractile properties of α-tropomyosin (TPM1) mutants associated with inherited cardiomyopathies.

J Biol Chem 2015 Mar 29;290(11):7003-15. Epub 2014 Dec 29.

From the Institute for Stem Cell Biology and Regenerative Medicine, Bangalore 560065, India, the McLaughlin Research Institute, Great Falls, Montana 59405

The most frequent known causes of primary cardiomyopathies are mutations in the genes encoding sarcomeric proteins. Among those are 30 single-residue mutations in TPM1, the gene encoding α-tropomyosin. We examined seven mutant tropomyosins, E62Q, D84N, I172T, L185R, S215L, D230N, and M281T, that were chosen based on their clinical severity and locations along the molecule. The goal of our study was to determine how the biochemical characteristics of each of these mutant proteins are altered, which in turn could provide a structural rationale for treatment of the cardiomyopathies they produce. Measurements of Ca(2+) sensitivity of human β-cardiac myosin ATPase activity are consistent with the hypothesis that hypertrophic cardiomyopathies are hypersensitive to Ca(2+) activation, and dilated cardiomyopathies are hyposensitive. We also report correlations between ATPase activity at maximum Ca(2+) concentrations and conformational changes in TnC measured using a fluorescent probe, which provide evidence that different substitutions perturb the structure of the regulatory complex in different ways. Moreover, we observed changes in protein stability and protein-protein interactions in these mutants. Our results suggest multiple mechanistic pathways to hypertrophic and dilated cardiomyopathies. Finally, we examined a computationally designed mutant, E181K, that is hypersensitive, confirming predictions derived from in silico structural analysis.
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http://dx.doi.org/10.1074/jbc.M114.596676DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4358124PMC
March 2015

Hypertrophic and dilated cardiomyopathy: four decades of basic research on muscle lead to potential therapeutic approaches to these devastating genetic diseases.

Authors:
James A Spudich

Biophys J 2014 Mar;106(6):1236-49

Department of Biochemistry, Stanford University School of Medicine, Stanford, California. Electronic address:

With the advent of technologies to obtain the complete sequence of the human genome in a cost-effective manner, this decade and those to come will see an exponential increase in our understanding of the underlying genetics that lead to human disease. And where we have a deep understanding of the biochemical and biophysical basis of the machineries and pathways involved in those genetic changes, there are great hopes for the development of modern therapeutics that specifically target the actual machinery and pathways altered by individual mutations. Prime examples of such a genetic disease are those classes of hypertrophic and dilated cardiomyopathy that result from single amino-acid substitutions in one of several of the proteins that make up the cardiac sarcomere or from the truncation of myosin binding protein C. Hypertrophic cardiomyopathy alone affects ∼1 in 500 individuals, and it is the leading cause of sudden cardiac death in young adults. Here I describe approaches to understand the molecular basis of the alterations in power output that result from these mutations. Small molecules binding to the mutant sarcomeric protein complex should be able to mitigate the effects of hypertrophic and dilated cardiomyopathy mutations at their sources, leading to possible new therapeutic approaches for these genetic diseases.
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http://dx.doi.org/10.1016/j.bpj.2014.02.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3985504PMC
March 2014

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.

PLoS One 2013 18;8(12):e83403. Epub 2013 Dec 18.

Department of Biochemistry, Stanford University School of Medicine, Stanford, California, United States of America ; Department of Pediatrics (Cardiology), Stanford University School of Medicine, Stanford, California, United States of America.

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.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0083403PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867432PMC
October 2014

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

Proc Natl Acad Sci U S A 2013 Jul 24;110(31):12607-12. Epub 2013 Jun 24.

Department of Biochemistry, Stanford University School of Medicine, Stanford, CA 94305, USA.

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.
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http://dx.doi.org/10.1073/pnas.1309493110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3732973PMC
July 2013
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