Mutant Myosin Research Articles

Dynamics of hydraulic and contractile wave-mediated fluid transport during Drosophila oogenesis

From insects to mice, oocytes develop within cysts alongside nurse-like sister germ cells. Prior to fertilization, the nurse cells' cytoplasmic contents are transported into the oocyte, which grows as its sister cells regress and die. Although critical for fertility, the biological and physical mechanisms underlying this transport process are poorly understood. Here, we combined live imaging of germline cysts, genetic perturbations, and mathematical modeling to investigate the dynamics and mechanisms that enable directional and complete cytoplasmic transport in Drosophila melanogaster egg chambers. We discovered that during "nurse cell (NC) dumping" most cytoplasm is transported into the oocyte independently of changes in myosin-II contractility, with dynamics instead explained by an effective Young-Laplace law, suggesting hydraulic transport induced by baseline cell-surface tension. A minimal flow-network model inspired by the famous two-balloon experiment and motivated by genetic analysis of a myosin mutant correctly predicts the directionality, intercellular pattern, and time scale of transport. Long thought to trigger transport through "squeezing," changes in actomyosin contractility are required only once NC volume has become comparable to nuclear volume, in the form of surface contractile waves that drive NC dumping to completion. Our work thus demonstrates how biological and physical mechanisms cooperate to enable a critical developmental process that, until now, was thought to be mainly biochemically regulated.

Molecular Mechanism of Force Generation by the Actin-Myosin Complex During Cardiac Muscle Contraction

Cardiac muscle contraction is performed by arrays of contractile proteins in the sarcomere. Serious heart diseases, such as cardiomyopathy, can often be results of mutations in myosin and actin, which slide along each other to generate force using the energy from ATP hydrolysis. All-atom molecular dynamics (MD) simulations have been growing its power in predicting protein structure-function relationships. However, such approaches are limited in the study of the myosin crossbridge cycle owing to the slow timescale as well as the lack of high-resolution structures of various intermediate states for the human cardiac myosin-actin complex. Here, combining comparative modeling and enhanced sampling MD simulations, we characterize how the myosin-actin interactions in human are influenced by the myosin functional states. By incorporating structural information from multiple templates, our comparative modeling provides conformational ensembles for different myosin-actin states, which enable us to efficiently sample the energy landscape using Gaussian accelerated MD simulations. Our results reveal novel interactions between key myosin loops and actin, which are consistent with recent cryo-EM density data. Furthermore, we find that the myosin cleft motion is impacted by the ATP binding site dynamics and that ADP release is coupled to the lever arm swing. Our approach demonstrates the potential to link genotype and molecular structures to the physical properties of the sarcomere.

Near-Atomic Structure of the 10S form of Myosin II: Implications for Inhibition, Activation and Disease

Myosin II is the molecular motor that powers contractility in muscle and nonmuscle cells. The two-headed molecule can exist in two conformations: compact (10S sedimentation coefficient) and extended (6S). The extended conformation assembles into filaments, which pull on actin to generate muscle contraction and cell motility. In the 10S structure, the tail is folded into three segments and the heads bend back and interact with each other and the tail, switching off ATPase activity, actin-activation and filament assembly. This energy-conserving, storage form can be activated by phosphorylation of its regulatory light chains (RLC) to form functional filaments as needed. We have solved the structure of smooth muscle 10S myosin to 4.3 Å resolution by cryo-EM, revealing near-atomic insights into its structure and inhibitory function (Yang et al., Nature, in press). The resulting refined atomic model shows clear secondary structure (α-helices, coiled-coil), with side-chain detail in some regions. The motor domains have the same (ADP.Pi) conformation, but their regulatory domains differ, allowing the two heads to meet asymmetrically. The coiled-coil shows considerable variations in pitch along its length. The regulatory domain backbone remains mostly helical as it joins the tail, with little apparent melting. The model reveals that the heads are prevented from binding to actin by steric clashes involving the tail and the other head, while their ATPase activity is impeded by constraints on movement of their converter domains. The positively charged N-terminal region of the RLC interacts with negative charge on the tail near the RLC phosphorylation site, suggesting a simple mechanism for activation. Disease-causing mutations in smooth and nonmuscle myosin II occur close to sites of head interaction with the tail, suggesting that interference with the 10S structure may underlie such diseases.

Simulating Cardiac Muscle Mutations in a Compliant Lattice with Commonly Used Assays

Cardiac muscle cells consist of a highly organized filament lattice with repetitive structure. Both inter- and intra-filament interactions control muscle activation and relaxation, and even small perturbations of these interactions can result in the development of cardiomyopathies. Building on an existing spatially explicit computational model of muscle contraction at the half-sarcomere level, we simulated different mutations by changing model parameters using published biochemical or biomechanical data. Importantly, we added the ability to specify profiles of protein properties and their frequency (penetrance) within the model. We assigned the mutant properties to a portion of the proteins, with the remainder retaining wildtype properties. The current model consists of 8 thin filaments, with a four state cardiac troponin-tropomyosin regulation cycle, determining accessibility of actin binding sites for myosin motor heads arising from the 4 thick filaments. Upon binding, the myosin motors follow a three-state model of the crossbridge cycle to generate axial tension. We simulated the cardiac troponin c (cTnC) mutation L48Q, which has increased calcium binding affinity, and the non-functional cTnC mutation D65A, which prevents calcium from binding at site II (contraction trigger site). Additionally, we co-varied cross-bridge cycle transition rates, increasing the attachment rate and decreasing the detachment rates to simulate the effect of myosin mutations, to study how this affected contraction with different dosages of L48Q. We simulated isometric twitch force, the force-pCa relationship, and filament material properties assays. For the isometric twitches, we specified a calcium transient derived from published intra-lattice measurements. Our results show emergent contractile dynamics similar to the respective experimental phenotypes. L48Q, with increasing penetrance, increases calcium sensitivity and linearly increases maximum tension, as well as time at maximum tension. D65A decreases maximum tension, and our myosin treatment increases maximum tension, and slows relaxation.

Embryonic Myosin Mutations T178I and R672C Develop Mechanical Dysfunction When Expressed in Hipsc Derived Skeletal Myotubes

Embryonic Myosin Mutations T178I and R672C Develop Mechanical Dysfunction When Expressed in Hipsc Derived Skeletal Myotubes

Prolonged myosin binding increases muscle stiffness in Drosophila models of Freeman-Sheldon syndrome

Freeman-Sheldon syndrome (FSS) is characterized by congenital contractures resulting from dominant point mutations in the embryonic isoform of muscle myosin. To investigate its disease mechanism, we used Drosophila models expressing FSS myosin mutations Y583S or T178I in their flight and jump muscles. We isolated these muscles from heterozygous mutant Drosophila and performed skinned fiber mechanics. The most striking mechanical alteration was an increase in active muscle stiffness. Y583S/+ and T178I/+ fibers’ elastic moduli increased 70 and 77%, respectively. Increased stiffness contributed to decreased power generation, 49 and 66%, as a result of increased work absorbed during the lengthening portion of the contractile cycle. Slower muscle kinetics also contributed to the mutant phenotype, as shown by 17 and 32% decreases in optimal frequency for power generation, and 27 and 41% slower muscle apparent rate constant 2πb. Combined with previous measurements of slower in vitro actin motility, our results suggest a rate reduction of at least one strongly bound cross-bridge cycle transition that increases the time myosin spends strongly bound to actin, ton. Increased ton was further supported by decreased ATP affinity and a 16% slowing of jump muscle relaxation rate in T178I heterozygotes. Impaired muscle function caused diminished flight and jump ability of Y583S/+ and T178I/+ Drosophila. Based on our results, assuming that our model system mimics human skeletal muscle, we propose that one mechanism driving FSS is elevated muscle stiffness arising from prolonged ton in developing muscle fibers.

Synthetic thick filaments: A new avenue for better understanding the myosin super-relaxed state in healthy, diseased, and mavacamten-treated cardiac systems

A hallmark feature of myosin-II is that it can spontaneously self-assemble into bipolar synthetic thick filaments (STFs) in low-ionic-strength buffers, thereby serving as a reconstituted in vitro model for muscle thick filaments. Although these STFs have been extensively used for structural characterization, their functional evaluation has been limited. In this report, we show that myosins in STFs mirror the more electrostatic and cooperative interactions that underlie the energy-sparing super-relaxed (SRX) state, which are not seen using shorter myosin subfragments, heavy meromyosin (HMM) and myosin subfragment 1 (S1). Using these STFs, we show several pathophysiological insults in hypertrophic cardiomyopathy, including the R403Q myosin mutation, phosphorylation of myosin light chains, and an increased ADP:ATP ratio, destabilize the SRX population. Furthermore, WT myosin containing STFs, but not S1, HMM, or STFs-containing R403Q myosin, recapitulated the ADP-induced destabilization of the SRX state. Studies involving a clinical-stage small-molecule inhibitor, mavacamten, showed that it is more effective in not only increasing myosin SRX population in STFs than in S1 or HMM but also in increasing myosin SRX population equally well in STFs made of healthy and disease-causing R403Q myosin. Importantly, we also found that pathophysiological perturbations such as elevated ADP concentration weakens mavacamten’s ability to increase the myosin SRX population, suggesting that mavacamten-bound myosin heads are not permanently protected in the SRX state but can be recruited into action. These findings collectively emphasize that STFs serve as a valuable tool to provide novel insights into the myosin SRX state in healthy, diseased, and therapeutic conditions.

External Mechanical Cues Reveal a Katanin-Independent Mechanism behind Auxin-Mediated Tissue Bending in Plants.

Tissue folding is a central building block of plant and animal morphogenesis. In dicotyledonous plants, hypocotyl folds to form hooks after seedling germination that protects their aerial stem cell niche during emergence from soil. Auxin response factors and auxin transport are reported to play a key role in this process. Here, we show that the microtubule-severing enzyme katanin contributes to hook formation. However, by exposing hypocotyls to external mechanical cues mimicking the natural soil environment, we reveal that auxin response factors ARF7/ARF19, auxin influx carriers, and katanin are dispensable for apical hook formation, indicating that these factors primarily play the role of catalyzers of tissue bending in the absence of external mechanical cues. Instead, our results reveal the key roles of the non-canonical TMK-mediated auxin pathway, PIN efflux carriers, and cellulose microfibrils as components of the core pathway behind hook formation in the presence or absence of external mechanical cues.

Impaired muscle morphology in a Drosophila model of myosin storage myopathy was supressed by overexpression of an E3 ubiquitin ligase.

ABSTRACTMyosin is vital for body movement and heart contractility. Mutations in MYH7, encoding slow/β-cardiac myosin heavy chain, are an important cause of hypertrophic and dilated cardiomyopathy, as well as skeletal muscle disease. A dominant missense mutation (R1845W) in MYH7 has been reported in several unrelated cases of myosin storage myopathy. We have developed a Drosophila model for a myosin storage myopathy in order to investigate the dose-dependent mechanisms underlying the pathological roles of the R1845W mutation. This study shows that a higher expression level of the mutated allele is concomitant with severe impairment of muscle function and progressively disrupted muscle morphology. The impaired muscle morphology associated with the mutant allele was suppressed by expression of Thin (herein referred to as Abba), an E3 ubiquitin ligase. This Drosophila model recapitulates pathological features seen in myopathy patients with the R1845W mutation and severe ultrastructural abnormalities, including extensive loss of thick filaments with selective A-band loss, and preservation of I-band and Z-disks were observed in indirect flight muscles of flies with exclusive expression of mutant myosin. Furthermore, the impaired muscle morphology associated with the mutant allele was suppressed by expression of Abba. These findings suggest that modification of the ubiquitin proteasome system may be beneficial in myosin storage myopathy by reducing the impact of MYH7 mutation in patients.

High-Resolution Cryo-EM Structure of the Cardiac Actomyosin Complex

Heart contraction depends on a complicated array of interactions between sarcomeric proteins required to convert chemical energy into mechanical force. Cyclic interactions between actin and myosin molecules, controlled by troponin and tropomyosin, generate the sliding force between the actin-based thin and myosin-based thick filaments. Alterations in this sophisticated system due to missense mutations can lead to cardiovascular diseases. Numerous structural studies proposed pathological mechanisms of missense mutations at the myosin-myosin, actin-tropomyosin, and tropomyosin-troponin interfaces. However, despite the central role of actomyosin interactions a detailed structural description of the cardiac actomyosin interface remained unknown. Here, we report a cryo-EM structure of a cardiac actomyosin complex at 3.8Å resolution. The structure reveals the molecular basis of cardiac diseases caused by missense mutations in myosin and actin proteins.

Drosophila myosin mutants model the disparate severity of type 1 and type 2B distal arthrogryposis and indicate an enhanced actin affinity mechanism

BackgroundDistal arthrogryposis (DA) is a group of autosomal dominant skeletal muscle diseases characterized by congenital contractures of distal limb joints. The most common cause of DA is a mutation of the embryonic myosin heavy chain gene, MYH3. Human phenotypes of DA are divided into the weakest form–DA1, a moderately severe form–DA2B (Sheldon-Hall Syndrome), and a severe DA disorder–DA2A (Freeman-Sheldon Syndrome). As models of DA1 and DA2B do not exist, their disease mechanisms are poorly understood.MethodsWe produced the first models of myosin-based DA1 (F437I) and DA2B (A234T) using transgenic Drosophila melanogaster and performed an integrative analysis of the effects of the mutations. Assessments included lifespan, locomotion, ultrastructural analysis, muscle mechanics, ATPase activity, in vitro motility, and protein modeling.ResultsWe observed significant defects in DA1 and DA2B Drosophila flight and jump ability, as well as myofibril assembly and stability, with homozygotes displaying more severe phenotypes than heterozygotes. Notably, DA2B flies showed dramatically stronger phenotypic defects compared to DA1 flies, mirroring the human condition. Mechanical studies of indirect flight muscle fibers from DA1 heterozygotes revealed reduced power output along with increased stiffness and force production, compared to wild-type controls. Further, isolated DA1 myosin showed significantly reduced myosin ATPase activity and in vitro actin filament motility. These data in conjunction with our sinusoidal analysis of fibers suggest prolonged myosin binding to actin and a slowed step associated with Pi release and/or the power stroke. Our results are supported by molecular modeling studies, which indicate that the F437I and A234T mutations affect specific amino acid residue interactions within the myosin motor domain that may alter interaction with actin and nucleotide.ConclusionsThe allele-specific ultrastructural and locomotory defects in our Drosophila DA1 and DA2B models are concordant with the differential severity of the human diseases. Further, the mechanical and biochemical defects engendered by the DA1 mutation reveal that power production, fiber stiffness, and nucleotide handling are aberrant in F437I muscle and myosin. The defects observed in our DA1 and DA2B Drosophila models provide insight into DA phenotypes in humans, suggesting that contractures arise from prolonged actomyosin interactions.

Manipulating Levels of Stress‐Response Proteins in a Drosophila Model of Myosin‐Based Inclusion Body Myopathy 3 Worsens Muscle Dysfunction

Utilizing a Drosophila model of inclusion body myopathy 3 (IBM‐3), we identified a subset of stress‐response proteins essential for skeletal muscle proteostasis in both health and disease. Inclusion body myopathy 3 (IBM‐3) is a rare, dominant skeletal muscle disease caused by an E706K substitution in the SH1 helix of myosin heavy chain IIa. We produced a model of the human disease in the indirect flight muscle (IFM) of Drosophila. As in the human disease, progressive degradation occurs in muscle expressing the IBM‐3 mutant myosin. Homozygotes assemble defective sarcomeres that accumulate thermally unstable myosin and autophagic vesicles. To further investigate the mechanism of pathogenesis in IBM‐3 muscle fibers, we extracted insoluble proteins from IFM of young and old homozygotes and identified aggregated proteins within inclusions. Quantitative iTRAQ proteomics defined 18 proteins with >1.5‐fold difference in relative abundance at both ages in mutants vs. controls. Two of 6 proteins with increased levels in IBM‐3 have small heat shock protein domains (Hsp20, CG7409), while the 12 proteins with decreased levels include Hsp23, Hsp60, and Trim32 (abba/thin), which has ubiquitin‐protein transferase activity. Using the GAL4‐UAS system for over‐expression or knock down (RNAi), we tested the role of these proteins in wild‐type muscle and determined how altering expression affects the IBM‐3 heterozygote phenotype. While knockdown of Hsp20, Hsp23 or CG7409 yielded normal flight in wild type, Hsp23 or CG7409 over‐expression resulted in age‐dependent reduction in flight ability. Knockdown or over‐expression of these chaperones typically exacerbated the IBM‐3/+ phenotype. As expected, Trim32 knockdown eliminated flight in wild‐type or IBM‐3/+ flies and correlated with less organized, thinner myofibrils. Overexpression of Trim32 also eliminated flight and yielded deteriorating myofibrils that hypercontracted by 3 weeks of age. Remarkably, electron microscopy shows the ultrastructure of this phenotype in IBM‐3/+ resembles the dramatic destruction of myofibrils observed in IBM‐3 homozygotes. Overall, each of the stress response proteins assessed is critical for proper muscle development, performance, or maintenance. Altering levels of these key proteins in muscle fibers challenged with the IBM‐3/+ phenotype has detrimental effects. Our findings add to evidence that expression levels of stress response proteins modulate alternative pathways to respond to challenges to proteostasis.Support or Funding InformationThis work was supported by grants from the Muscular Dystrophy Association (MDA217900)and the National Institutes of Health (NIGMS R37 32443) to S.I.B. The authors acknowledgethe Bloomington Drosophila Stock Center (NIH P40OD018537), the Vienna DrosophilaResource Center and Dr. Richard Cripps (SDSU) for Drosophila stocks.

Uncovering the Molecular and Structural Basis of Hypertrophic Cardiomyopathy-Causing Mutations in Myosin and Myosin Binding Protein-C

Uncovering the Molecular and Structural Basis of Hypertrophic Cardiomyopathy-Causing Mutations in Myosin and Myosin Binding Protein-C

Use of Cells from Remotely Collected Urine to Generate Human Induced Pluripotent Stem Cells and Myofibers that Recapitulate Unconventional Myopathies

Mutations in embryonic myosin (MYH3) disrupt the function of skeletal muscle early in fetal development resulting in congenital contractures of multiple body areas, most commonly the hands and feet. In the most severe condition, Freeman-Sheldon Syndrome (FSS), muscles of the face and back are also involved. Study of the mechanism(s) underlying formation of contractures, as well as development of targeted therapeutics for contractures, are severely limited by the difficulty of obtaining skeletal muscle from affected individuals. Innovative approaches are therefore needed to model disease mechanisms and test therapeutics that could improve clinical outcomes. We show that urine collected at home can be used to harvest cells for reprogramming into induced pluripotent stem cells (iPSCs) and then differentiated into spontaneously contracting skeletal muscle. We identified individuals (ages 12-50 years) with the most common MYH3 mutations, p.R672C, p.R672H, and p.T178I, in underlying FSS. Kits were sent to each person to collect their first morning-voided urine and this sample was returned to us overnight. Somatic cells were extracted from the urine and reprogrammed using a CytoTune 2.0 Sendai kit. The iPSC lines have a normal karyotype, express common pluripotency markers, and maintain the original MYH3 mutations. Cells were successfully reprogrammed in all three persons tested. Preliminary functional studies using individual contractile organelles (myofibrils) show that mutant cells have reduced contractile force and prolonged relaxation times. Myofibers (cells) loaded with Fluo4 AM calcium dye displayed delayed calcium reuptake during relaxation. These findings validate our approach to reprogramming and muscle differentiation, yielding myogenic cells that recapitulate the disease phenotype identified in myofibers sampled from affected individuals. Moreover, these myogenic cells are now available for development and testing of therapeutics.

Impact of Regulatory Light Chain Mutation (K104E) on the Atpase and Motor Properties of Human Cardiac Myosin

Mutations in the cardiac myosin heavy chain (MYH7) as well as the essential (MYL3) and regulatory (MYL2) light chains are known to lead to cardiomyopathies with variable phenotypes, including hypertrophic (HCM) and dilated (DCM) cardiomyopathy. We investigated the impact of the HCM-associated regulatory light chain (RLC) mutation (K104E) on myosin ATPase kinetics and in vitro motility. We expressed and purified human cardiac myosin subfragment 1 (M2B-S1) and exchanged on the human ventricular RLC, with the mutant or WT RLC. A mouse model of K104E was found to cause cardiac hypertrophy, fibrosis, and diastolic dysfunction in older animals suggesting a slow development of HCM, while variable penetrance of the mutation in human populations suggest the impact of K104E may be subtle. We found that maximum actin-activated ATPase activity and the actin-dependence of ATPase were similar in K104E and WT. Actin gliding velocities in the in vitro motility assay were slightly increased in K104E. We found that the ADP release rate constant in the presence of actin also was slightly increased in K104E compared to WT. Our results suggest that HCM-associated RLC mutations may only have a minor impact on unloaded ATPase kinetics and in vitro actin gliding, which may suggest other factors could be altered by the mutation such as mechanical load and myosin regulation. Future experiments will address the impact of the mutation in mouse cardiac vs. human cardiac myosin as well as the impact of the mutation on load-dependent motility and the off state (super-relaxed state) of cardiac myosin.

Study of Hcm Causing β-Cardiac Myosin Mutations Located at Different Structurally Significant Regions of the Myosin-Head

β-cardiac myosin dwells in an equilibrium between an actin-interacting ‘on’ state and an inactive folded-back ‘off’ state. During actin-interaction, myosin hydrolyzes ATP and undergoes conformational changes that move the thin filament with respect to the thick filament, resulting in cardiac contraction. ∼35% of heritable hypertrophic cardiomyopathy has been linked to β-cardiac myosin mutations. Although the myosin motor is highly allosteric, different regions of the motor serve distinct functions. As myosin heads interact with the actin, an ATP molecule gets hydrolyzed at the ATP-binding site (p-loop), and the converter domain and relay helix bring about required cyclic-conformational changes. Throughout the whole process the 7-stranded β-sheet at the core of myosin-head structure provides required rigidity to transfer force. Residues in the mesa and converter regions in the myosin-head, and residues in the proximal part of myosin-tail control the equilibrium between the ‘on’ and ‘off’ states. We plan to investigate how pathological β-cardiac myosin mutations — Y115H (β-sheet region), R169G (mesa region/near p-loop), E497D (relay helix), E536D (actin interacting/mesa region), and I702N (near converter domain) — alter function by performing ensemble and single molecule measurements. We prepared two new two-headed human β-cardiac constructs having different length of heptad units in the tail region for our test. One of the constructs has fifteen heptad repeats (15-hep) and is designed to support head-tail interaction in the “off” state, while the other construct has just eight heptads and is predicted to lack the head-tail interaction sites. Preliminary experiments show that the 15-hep, but not the 8-hep, construct showed a significant population in the super relaxed state by a single ATP turnover assay, consistent with only the 15-hep forming the off-state. Both construct show similar single molecule force measurements, as expected.

Allele-Selective Knockdown of MYH7 Using Antisense Oligonucleotides

Hundreds of dominant-negative myosin mutations have been identified that lead to hypertrophic cardiomyopathy, and the biomechanical link between mutation and disease is heterogeneous across this patient population. To increase the therapeutic feasibility of treating this diverse genetic population, we investigated the ability of locked nucleic acid (LNA)-modified antisense oligonucleotides (ASOs) to selectively knock down mutant myosin transcripts by targeting single-nucleotide polymorphisms (SNPs) that were found to be common in the myosin heavy chain 7 (MYH7) gene. We identified three SNPs in MYH7 and designed ASO libraries to selectively target either the reference or alternate MYH7 sequence. We identified ASOs that selectively knocked down either the reference or alternate allele at all three SNP regions. We also show allele-selective knockdown in a mouse model that was humanized on one allele. These results suggest that SNP-targeting ASOs are a promising therapeutic modality for treating cardiac pathology.

Dilated cardiomyopathy mutation in the converter domain of human cardiac myosin alters motor activity and response to omecamtiv mecarbil

We investigated a dilated cardiomyopathy (DCM) mutation (F764L) in human β-cardiac myosin by determining its motor properties in the presence and absence of the heart failure drug omecamtive mecarbil (OM). The mutation is located in the converter domain, a key region of communication between the catalytic motor and lever arm in myosins, and is nearby but not directly in the OM-binding site. We expressed and purified human β-cardiac myosin subfragment 1 (M2β-S1) containing the F764L mutation, and compared it to WT with in vitro motility as well as steady-state and transient kinetics measurements. In the absence of OM we demonstrate that the F764L mutation does not significantly change maximum actin-activated ATPase activity but slows actin sliding velocity (15%) and the actomyosin ADP release rate constant (25%). The transient kinetic analysis without OM demonstrates that F764L has a similar duty ratio as WT in unloaded conditions. OM is known to enhance force generation in cardiac muscle while it inhibits the myosin power stroke and enhances actin-attachment duration. We found that OM has a reduced impact on F764L ATPase and sliding velocity compared with WT. Specifically, the EC50 for OM induced inhibition of in vitro motility was 3-fold weaker in F764L. Also, OM reduces maximum actin-activated ATPase 2-fold in F764L, compared with 4-fold with WT. Overall, our results suggest that F764L attenuates the impact of OM on actin-attachment duration and/or the power stroke. Our work highlights the importance of mutation-specific considerations when pursuing small molecule therapies for cardiomyopathies.

Cellular defects resulting from disease-related myosin II mutations in Drosophila

The nonmuscle myosin II motor protein produces forces that are essential to driving the cell movements and cell shape changes that generate tissue structure. Mutations in myosin II that are associated with human diseases are predicted to disrupt critical aspects of myosin function, but the mechanisms that translate altered myosin activity into specific changes in tissue organization and physiology are not well understood. Here we use the Drosophila embryo to model human disease mutations that affect myosin motor activity. Using in vivo imaging and biophysical analysis, we show that engineering human MYH9-related disease mutations into Drosophila myosin II produces motors with altered organization and dynamics that fail to drive rapid cell movements, resulting in defects in epithelial morphogenesis. In embryos that express the Drosophila myosin motor variants R707C or N98K and have reduced levels of wild-type myosin, myosin motors are correctly planar polarized and generate anisotropic contractile tension in the tissue. However, expression of these motor variants is associated with a cellular-scale reduction in the speed of cell intercalation, resulting in a failure to promote full elongation of the body axis. In addition, these myosin motor variants display slowed turnover and aberrant aggregation at the cell cortex, indicating that mutations in the motor domain influence mesoscale properties of myosin organization and dynamics. These results demonstrate that disease-associated mutations in the myosin II motor domain disrupt specific aspects of myosin localization and activity during cell intercalation, linking molecular changes in myosin activity to defects in tissue morphogenesis.

Recessive MYH7-related myopathy in two families

Myopathies due to recessive MYH7 mutations are exceedingly rare, reported in only two families to date. We describe three patients from two families (from Australia and the UK) with a myopathy caused by recessive mutations in MYH7. The Australian family was homozygous for a c.5134C > T, p.Arg1712Trp mutation, whilst the UK patient was compound heterozygous for a truncating (c.4699C > T; p.Gln1567*) and a missense variant (c.4664A > G; p.Glu1555Gly). All three patients shared key clinical features, including infancy/childhood onset, pronounced axial/proximal weakness, spinal rigidity, severe scoliosis, and normal cardiac function. There was progressive respiratory impairment necessitating non-invasive ventilation despite preserved ambulation, a combination of features often seen in SEPN1- or NEB-related myopathies. On biopsy, the Australian proband showed classical myosin storage myopathy features, while the UK patient showed multi-minicore like areas. To establish pathogenicity of the Arg1712Trp mutation, we expressed mutant MYH7 protein in COS-7 cells, observing abnormal mutant myosin aggregation compared to wild-type. We describe skinned myofiber studies of patient muscle and hypertrophy of type II myofibers, which may be a compensatory mechanism. In summary, we have expanded the phenotype of ultra-rare recessive MYH7 disease, and provide novel insights into associated changes in muscle physiology.