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.

The R249Q hypertrophic cardiomyopathy myosin mutation decreases contractility in Drosophila by impeding force production.

KEY POINTS: Hypertrophic cardiomyopathy (HCM) is a genetic disease that causes thickening of the heart's ventricular walls and is a leading cause of sudden cardiac death. HCM is caused by missense mutations in muscle proteins including myosin, but how these mutations alter muscle mechanical performance in largely unknown. We investigated the disease mechanism for HCM myosin mutation R249Q by expressing it in the indirect flight muscle of Drosophila melanogaster and measuring alterations to muscle and flight performance. Muscle mechanical analysis revealed R249Q decreased muscle power production due to slower muscle kinetics and decreased force production; force production was reduced because fewer mutant myosin cross-bridges were bound simultaneously to actin. This work does not support the commonly proposed hypothesis that myosin HCM mutations increase muscle contractility, or causes a gain in function; instead, it suggests that for some myosin HCM mutations, hypertrophy is a compensation for decreased contractility. ABSTRACT: Hypertrophic cardiomyopathy (HCM) is an inherited disease that causes thickening of the heart's ventricular walls. A generally accepted hypothesis for this phenotype is that myosin heavy chain HCM mutations increase muscle contractility. To test this hypothesis, we expressed an HCM myosin mutation, R249Q, in Drosophila indirect flight muscle (IFM) and assessed myofibril structure, skinned fibre mechanical properties, and flight ability. Mechanics experiments were performed on fibres dissected from 2-h-old adult flies, prior to degradation of IFM myofilament structure, which started at 2 days old and increased with age. Homozygous and heterozygous R249Q fibres showed decreased maximum power generation by 67% and 44%, respectively. Decreases in force and work and slower overall muscle kinetics caused homozygous fibres to produce less power. While heterozygous fibres showed no overall slowing of muscle kinetics, active force and work production dropped by 68% and 47%, respectively, which hindered power production. The muscle apparent rate constant 2πb decreased 33% for homozygous but increased for heterozygous fibres. The apparent rate constant 2πc was greater for homozygous fibres. This indicates that R249Q myosin is slowing attachment while speeding up detachment from actin, resulting in less time bound. Decreased IFM power output caused 43% and 33% decreases in Drosophila flight ability and 19% and 6% drops in wing beat frequency for homozygous and heterozygous flies, respectively. Overall, our results do not support the increased contractility hypothesis. Instead, our results suggest the ventricular hypertrophy for human R249Q mutation is a compensatory response to decreases in heart muscle power output.

Myosin storage myopathy mutations yield defective myosin filament assembly in vitro and disrupted myofibrillar structure and function in vivo.

Myosin storage myopathy (MSM) is a congenital skeletal muscle disorder caused by missense mutations in the ß-cardiac/slow skeletal muscle myosin heavy chain rod. It is characterized by subsarcolemmal accumulations of myosin that have a hyaline appearance. MSM mutations map near or within the assembly competence domain known to be crucial for thick filament formation. Drosophila MSM models were generated for comprehensive physiological, structural, and biochemical assessment of the mutations' consequences on muscle and myosin structure and function. L1793P, R1845W, and E1883K MSM mutant myosins were expressed in an indirect flight (IFM) and jump muscle myosin null background to study the effects of these variants without confounding influences from wild-type myosin. Mutant animals displayed highly compromised jump and flight ability, disrupted muscle proteostasis, and severely perturbed IFM structure. Electron microscopy revealed myofibrillar disarray and degeneration with hyaline-like inclusions. In vitro assembly assays demonstrated a decreased ability of mutant myosin to polymerize, with L1793P filaments exhibiting shorter lengths. In addition, limited proteolysis experiments showed a reduced stability of L1793P and E1883K filaments. We conclude that the disrupted hydropathy or charge of residues in the heptad repeat of the mutant myosin rods likely alters interactions that stabilize coiled-coil dimers and thick filaments, causing disruption in ordered myofibrillogenesis and/or myofibrillar integrity, and the consequent myosin aggregation. Our Drosophila models are the first to recapitulate the human MSM phenotype with ultrastructural inclusions, suggesting that the diminished ability of the mutant myosin to form stable thick filaments contributes to the dystrophic phenotype observed in afflicted subjects.

A Failure to Communicate: MYOSIN RESIDUES INVOLVED IN HYPERTROPHIC CARDIOMYOPATHY AFFECT INTER-DOMAIN INTERACTION.

Our molecular modeling studies suggest a charge-dependent interaction between residues Glu-497 in the relay domain and Arg-712 in the converter domain of human ß-cardiac myosin. To test the significance of this putative interaction, we generated transgenic Drosophila expressing indirect flight muscle myosin with charge reversal mutations in the relay (E496R) or converter (R713E). Each mutation yielded dramatic reductions in myosin Ca-ATPase activity (~80%) as well as in basal (~67%) and actin-activated (~84%) Mg-ATPase activity. E496R myosin-induced in vitro actin-sliding velocity was reduced by 71% and R713E myosin permitted no actin motility. Indirect flight muscles of late pupae from each mutant displayed disrupted myofibril assembly, with adults having severely abnormal myofibrils and no flight ability. To understand the molecular basis of these defects, we constructed a putative compensatory mutant that expresses myosin with both E496R and R713E. Intriguingly, ATPase values were restored to ~73% of wild-type and actin-sliding velocity increased to 40%. The double mutation suppresses myofibril assembly defects in pupal indirect flight muscles and dramatically reduces myofibril disruption in young adults. Although sarcomere organization is not sustained in older flies and flight ability is not restored in homozygotes, young heterozygotes fly well. Our results indicate that this charge-dependent interaction between the myosin relay and converter domains is essential to the mechanochemical cycle and sarcomere assembly. Furthermore, the same inter-domain interaction is disrupted when modeling human ß-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.

Mapping interactions between myosin relay and converter domains that power muscle function.

Intramolecular communication within myosin is essential for its function as motor, but the specific amino acid residue interactions required are unexplored within muscle cells. Using Drosophila melanogaster skeletal muscle myosin, we performed a novel in vivo molecular suppression analysis to define the importance of three relay loop amino acid residues (Ile(508), Asn(509), and Asp(511)) in communicating with converter domain residue Arg(759). We found that the N509K relay mutation suppressed defects in myosin ATPase, in vitro motility, myofibril stability, and muscle function associated with the R759E converter mutation. Through molecular modeling, we define a mechanism for this interaction and suggest why the I508K and D511K relay mutations fail to suppress R759E. Interestingly, I508K disabled motor function and myofibril assembly, suggesting that productive relay-converter interaction is essential for both processes. We conclude that the putative relay-converter interaction mediated by myosin residues 509 and 759 is critical for the biochemical and biophysical function of skeletal muscle myosin and the normal ultrastructural and mechanical properties of muscle.

Aging-affiliated post-translational modifications of skeletal muscle myosin affect biochemical properties, myofibril structure, muscle function, and proteostasis.

The molecular motor myosin is post-translationally modified in its globular head, its S2 hinge, and its thick filament domain during human skeletal muscle aging. To determine the importance of such modifications, we performed an integrative analysis of transgenic Drosophila melanogaster expressing myosin containing post-translational modification mimic mutations. We determined effects on muscle function, myofibril structure, and myosin biochemistry. Modifications in the homozygous state decreased jump muscle function by a third at 3 weeks of age and reduced indirect flight muscle function to negligible levels in young flies, with severe effects on flight muscle myofibril assembly and/or maintenance. Expression of mimic mutations in the heterozygous state or in a wild-type background yielded significant, but less severe, age-dependent effects upon flight muscle structure and function. Modification of the residue in the globular head disabled ATPase activity and in vitro actin filament motility, whereas the S2 hinge mutation reduced actin-activated ATPase activity by 30%. The rod modification diminished filament formation in vitro. The latter mutation also reduced proteostasis, as demonstrated by enhanced accumulation of polyubiquitinated proteins. Overall, we find that mutation of amino acids at sites that are chemically modified during human skeletal muscle aging can disrupt myosin ATPase, myosin filament formation, and/or proteostasis, providing a mechanistic basis for the observed muscle defects. We conclude that age-specific post-translational modifications present in human skeletal muscle are likely to act in a dominant fashion to affect muscle structure and function and may therefore be implicated in degeneration and dysfunction associated with sarcopenia.

Drosophila UNC-45 accumulates in embryonic blastoderm and in muscles, and is essential for muscle myosin stability.

UNC-45 is a chaperone that facilitates folding of myosin motor domains. We have used Drosophila melanogaster to investigate the role of UNC-45 in muscle development and function. Drosophila UNC-45 (dUNC-45) is expressed at all developmental stages. It colocalizes with non-muscle myosin in embryonic blastoderm of 2-hour-old embryos. At 14 hours, it accumulates most strongly in embryonic striated muscles, similarly to muscle myosin. dUNC-45 localizes to the Z-discs of sarcomeres in third instar larval body-wall muscles. We produced a dunc-45 mutant in which zygotic expression is disrupted. This results in nearly undetectable dUNC-45 levels in maturing embryos as well as late embryonic lethality. Muscle myosin accumulation is robust in dunc-45 mutant embryos at 14 hours. However, myosin is dramatically decreased in the body-wall muscles of 22-hour-old mutant embryos. Furthermore, electron microscopy showed only a few thick filaments and irregular thick-thin filament lattice spacing. The lethality, defective protein accumulation, and ultrastructural abnormalities are rescued with a wild-type dunc-45 transgene, indicating that the mutant phenotypes arise from the dUNC-45 deficiency. Overall, our data indicate that dUNC-45 is important for myosin accumulation and muscle function. Furthermore, our results suggest that dUNC-45 acts post-translationally for proper myosin folding and maturation.

Myosin Transducer Inter-Strand Communication Is Critical for Normal ATPase Activity and Myofibril Structure.

The R249Q mutation in human ß-cardiac myosin results in hypertrophic cardiomyopathy. We previously showed that inserting this mutation into Drosophila melanogaster indirect flight muscle myosin yields mechanical and locomotory defects. Here, we use transgenic Drosophila mutants to demonstrate that residue R249 serves as a critical communication link within myosin that controls both ATPase activity and myofibril integrity. R249 is located on a ß-strand of the central transducer of myosin, and our molecular modeling shows that it interacts via a salt bridge with D262 on the adjacent ß-strand. We find that disrupting this interaction via R249Q, R249D or D262R mutations reduces basal and actin-activated ATPase activity, actin in vitro motility and flight muscle function. Further, the R249D mutation dramatically affects myofibril assembly, yielding abnormalities in sarcomere lengths, increased Z-line thickness and split myofibrils. These defects are exacerbated during aging. Re-establishing the ß-strand interaction via a R249D/D262R double mutation restores both basal ATPase activity and myofibril assembly, indicating that these properties are dependent upon transducer inter-strand communication. Thus, the transducer plays an important role in myosin function and myofibril architecture.

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

BACKGROUND: Distal 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. METHODS: We 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. RESULTS: We 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. CONCLUSIONS: The 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.

Reductions in ATPase activity, actin sliding velocity, and myofibril stability yield muscle dysfunction in Drosophila models of myosin-based Freeman-Sheldon syndrome.

Using Drosophila melanogaster, we created the first animal models for myosin-based Freeman-Sheldon syndrome (FSS), a dominant form of distal arthrogryposis defined by congenital facial and distal skeletal muscle contractures. Electron microscopy of homozygous mutant indirect flight muscles showed normal (Y583S) or altered (T178I, R672C) myofibril assembly followed by progressive disruption of the myofilament lattice. In contrast, all alleles permitted normal myofibril assembly in the heterozygous state but caused myofibrillar disruption during aging. The severity of myofibril defects in heterozygotes correlated with the level of flight impairment. Thus our Drosophila models mimic the human condition in that FSS mutations are dominant and display varied degrees of phenotypic severity. Molecular modeling indicates that the mutations disrupt communication between the nucleotide-binding site of myosin and its lever arm that drives force production. Each mutant myosin showed reduced in vitro actin sliding velocity, with the two more severe alleles significantly decreasing the catalytic efficiency of actin-activated ATP hydrolysis. The observed reductions in actin motility and catalytic efficiency may serve as the mechanistic basis of the progressive myofibrillar disarray observed in the Drosophila models as well as the prolonged contractile activity responsible for skeletal muscle contractures in FSS patients.

Alternative versions of the myosin relay domain differentially respond to load to influence Drosophila muscle kinetics.

We measured the influence of alternative versions of the Drosophila melanogaster myosin heavy chain relay domain on muscle mechanical properties. We exchanged relay domain regions (encoded by alternative versions of exon 9) between an embryonic (EMB) isoform and the indirect flight muscle isoform (IFI) of myosin. Previously, we observed no effect of exchanging the EMB relay domain region into the flight muscle isoform (IFI-9b) on in vitro actin motility velocity or solution ATPase measurements compared to IFI. However, in indirect flight muscle fibers, IFI-9b exhibited decreased maximum power generation (P(max)) and optimal frequency of power generation (f(max)) to 70% and 83% of IFI fiber values. The decrease in muscle performance reduced the flight ability and wing-beat frequency of IFI-9b Drosophila compared to IFI Drosophila. Previously, we found that exchanging the flight muscle specific relay domain into the EMB isoform (EMB-9a) prevented actin movement in the in vitro motility assay compared to EMB, which does support actin movement. However, in indirect flight muscle fibers EMB-9a was a highly effective motor, increasing P(max) and f(max) 2.5-fold and 1.4-fold, respectively, compared to fibers expressing EMB. We propose that the oscillatory load EMB-9a experiences in the muscle fiber reduces a high activation energy barrier between two strongly bound states of the cross-bridge cycle, thereby promoting cross-bridge cycling. The IFI relay domain's enhanced sensitivity to load increases cross-bridge kinetics, whereas the EMB version is less load-sensitive.

Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length.

Muscle myosin heavy chain (MHC) rod domains intertwine to form alpha-helical coiled-coil dimers; these subsequently multimerize into thick filaments via electrostatic interactions. The subfragment 2/light meromyosin "hinge" region of the MHC rod, located in the C-terminal third of heavy meromyosin, may form a less stable coiled-coil than flanking regions. Partial "melting" of this region has been proposed to result in a helix to random-coil transition. A portion of the Drosophila melanogaster MHC hinge is encoded by mutually exclusive alternative exons 15a and 15b, the use of which correlates with fast (hinge A) or slow (hinge B) muscle physiological properties. To test the functional significance of alternative hinge regions, we constructed transgenic fly lines in which fast muscle isovariant hinge A was switched for slow muscle hinge B in the MHC isoforms of indirect flight and jump muscles. Substitution of the slow muscle hinge B impaired flight ability, increased sarcomere lengths by approximately 13% and resulted in minor disruption to indirect flight muscle sarcomeric structure compared with a transgenic control. With age, residual flight ability decreased rapidly and myofibrils developed peripheral defects. Computational analysis indicates that hinge B has a greater coiled-coil propensity and thus reduced flexibility compared to hinge A. Intriguingly, the MHC rod with hinge B was approximately 5 nm longer than myosin with hinge A, consistent with the more rigid coiled-coil conformation predicted for hinge B. Our study demonstrates that hinge B cannot functionally substitute for hinge A in fast muscle types, likely as a result of differences in the molecular structure of the rod, subtle changes in myofibril structure and decreased ability to maintain sarcomere structure in indirect flight muscle myofibrils. Thus, alternative hinges are important in dictating the distinct functional properties of myosin isoforms and the muscles in which they are expressed.

Prolonged cross-bridge binding triggers muscle dysfunction in a Drosophila model of myosin-based hypertrophic cardiomyopathy.

K146N is a dominant mutation in human ß-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined how Drosophila muscle responds to this mutation and integratively analyzed the biochemical, physiological and mechanical foundations of the disease. ATPase assays, actin motility, and indirect flight muscle mechanics suggest at least two rate constants of the cross-bridge cycle are altered by the mutation: increased myosin attachment to actin and decreased detachment, yielding prolonged binding. This increases isometric force generation, but also resistive force and work absorption during cyclical contractions, resulting in decreased work, power output, flight ability and degeneration of flight muscle sarcomere morphology. Consistent with prolonged cross-bridge binding serving as the mechanistic basis of the disease and with human phenotypes, 146N/+ hearts are hypercontractile with increased tension generation periods, decreased diastolic/systolic diameters and myofibrillar disarray. This suggests that screening mutated Drosophila hearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

Profilin modulates sarcomeric organization and mediates cardiomyocyte hypertrophy.

AIMS: Heart failure is often preceded by cardiac hypertrophy, which is characterized by increased cell size, altered protein abundance, and actin cytoskeletal reorganization. Profilin is a well-conserved, ubiquitously expressed, multifunctional actin-binding protein, and its role in cardiomyocytes is largely unknown. Given its involvement in vascular hypertrophy, we aimed to test the hypothesis that profilin-1 is a key mediator of cardiomyocyte-specific hypertrophic remodelling. METHODS AND RESULTS: Profilin-1 was elevated in multiple mouse models of hypertrophy, and a cardiomyocyte-specific increase of profilin in Drosophila resulted in significantly larger heart tube dimensions. Moreover, adenovirus-mediated overexpression of profilin-1 in neonatal rat ventricular myocytes (NRVMs) induced a hypertrophic response, measured by increased myocyte size and gene expression. Profilin-1 silencing suppressed the response in NRVMs stimulated with phenylephrine or endothelin-1. Mechanistically, we found that profilin-1 regulates hypertrophy, in part, through activation of the ERK1/2 signalling cascade. Confocal microscopy showed that profilin localized to the Z-line of Drosophila myofibrils under normal conditions and accumulated near the M-line when overexpressed. Elevated profilin levels resulted in elongated sarcomeres, myofibrillar disorganization, and sarcomeric disarray, which correlated with impaired muscle function. CONCLUSION: Our results identify novel roles for profilin as an important mediator of cardiomyocyte hypertrophy. We show that overexpression of profilin is sufficient to induce cardiomyocyte hypertrophy and sarcomeric remodelling, and silencing of profilin attenuates the hypertrophic response.

Alternative relay and converter domains tune native muscle myosin isoform function in Drosophila.

Myosin isoforms help define muscle-specific contractile and structural properties. Alternative splicing of myosin heavy chain gene transcripts in Drosophila melanogaster yields muscle-specific isoforms and highlights alternative domains that fine-tune myosin function. To gain insight into how native myosin is tuned, we expressed three embryonic myosin isoforms in indirect flight muscles lacking endogenous myosin. These isoforms differ in their relay and/or converter domains. We analyzed isoform-specific ATPase activities, in vitro actin motility and myofibril structure/stability. We find that dorsal acute body wall muscle myosin (EMB-9c11d) shows a significant increase in MgATPase V(max) and actin sliding velocity, as well as abnormal myofibril assembly compared to cardioblast myosin (EMB-11d). These properties differ as a result of alternative exon-9-encoded relay domains that are hypothesized to communicate signals among the ATP-binding pocket, actin-binding site and the converter domain. Further, EMB-11d shows significantly reduced levels of basal Ca- and MgATPase as well as MgATPase V(max) compared to embryonic body wall muscle isoform (EMB) (expressed in a multitude of body wall muscles). EMB-11d also induces increased actin sliding velocity and stabilizes myofibril structure compared to EMB. These differences arise from exon-11-encoded alternative converter domains that are proposed to reposition the lever arm during the power and recovery strokes. We conclude that relay and converter domains of native myosin isoforms fine-tune ATPase activity, actin motility and muscle ultrastructure. This verifies and extends previous studies with chimeric molecules and indicates that interactions of the relay and converter during the contractile cycle are key to myosin-isoform-specific kinetic and mechanical functions.

Expression of the inclusion body myopathy 3 mutation in Drosophila depresses myosin function and stability and recapitulates muscle inclusions and weakness.

Hereditary myosin myopathies are characterized by variable clinical features. Inclusion body myopathy 3 (IBM-3) is an autosomal dominant disease associated with a missense mutation (E706K) in the myosin heavy chain IIa gene. Adult patients experience progressive muscle weakness. Biopsies reveal dystrophic changes, rimmed vacuoles with cytoplasmic inclusions, and focal disorganization of myofilaments. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E701K) indirect flight and jump muscles to establish a novel homozygous organism with homogeneous populations of fast IBM-3 myosin and muscle fibers. Flight and jump abilities were severely reduced in homozygotes. ATPase and actin sliding velocity of the mutant myosin were depressed >80% compared with wild-type myosin. Light scattering experiments and electron microscopy revealed that mutant myosin heads bear a dramatic propensity to collapse and aggregate. Thus E706K (E701K) myosin appears far more labile than wild-type myosin. Furthermore, mutant fly fibers exhibit ultrastructural hallmarks seen in patients, including cytoplasmic inclusions containing aberrant proteinaceous structures and disorganized muscle filaments. Our Drosophila model reveals the unambiguous consequences of the IBM-3 lesion on fast muscle myosin and fibers. The abnormalities observed in myosin function and muscle ultrastructure likely contribute to muscle weakness observed in our flies and patients.

Mutating the converter-relay interface of Drosophila myosin perturbs ATPase activity, actin motility, myofibril stability and flight ability.

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of the myosin molecular motor from Drosophila melanogaster indirect flight muscle. During the myosin mechanochemical cycle, ATP-induced twisting of the relay loop is hypothesized to reposition the converter, resulting in cocking of the contiguous lever arm into the pre-power stroke configuration. The subsequent movement of the lever arm through its power stroke generates muscle contraction by causing myosin heads to pull on actin filaments. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at a site of relay loop interaction. Molecular modeling suggests that the interface between the relay loop and converter domain of R759E myosin would be significantly disrupted during the mechanochemical cycle. The mutation depressed calcium as well as basal and actin-activated MgATPase (V(max)) by approximately 60% compared to wild-type myosin, but there is no change in apparent actin affinity (K(m)). While ATP or AMP-PNP (adenylyl-imidodiphosphate) binding to wild-type myosin subfragment-1 enhanced tryptophan fluorescence by approximately 15% or approximately 8%, respectively, enhancement does not occur in the mutant. This suggests that the mutation reduces lever arm movement. The mutation decreases in vitro motility of actin filaments by approximately 35%. Mutant pupal indirect flight muscles display normal myofibril assembly, myofibril shape, and double-hexagonal arrangement of thick and thin filaments. Two-day-old fibers have occasional "cracking" of the crystal-like array of myofilaments. Fibers from 1-week-old adults show more severe cracking and frayed myofibrils with some disruption of the myofilament lattice. Flight ability is reduced in 2-day-old flies compared to wild-type controls, with no upward mobility but some horizontal flight. In 1-week-old adults, flight capability is lost. Thus, altered myosin function permits myofibril assembly, but results in a progressive disruption of the myofilament lattice and flight ability. We conclude that R759 in the myosin converter domain is essential for normal ATPase activity, in vitro motility and locomotion. Our results provide the first mutational evidence that intramolecular signaling between the relay loop and converter domain is critical for myosin function both in vitro and in muscle.

Alternative relay domains of Drosophila melanogaster myosin differentially affect ATPase activity, in vitro motility, myofibril structure and muscle function.

The relay domain of myosin is hypothesized to function as a communication pathway between the nucleotide-binding site, actin-binding site and the converter domain. In Drosophila melanogaster, a single myosin heavy chain gene encodes three alternative relay domains. Exon 9a encodes the indirect flight muscle isoform (IFI) relay domain, whereas exon 9b encodes one of the embryonic body wall isoform (EMB) relay domains. To gain a better understanding of the function of the relay domain and the differences imparted by the IFI and the EMB versions, we constructed two transgenic Drosophila lines expressing chimeric myosin heavy chains in indirect flight muscles lacking endogenous myosin. One expresses the IFI relay domain in the EMB backbone (EMB-9a), while the second expresses the EMB relay domain in the IFI backbone (IFI-9b). Our studies reveal that the EMB relay domain is functionally equivalent to the IFI relay domain when it is substituted into IFI. Essentially no differences in ATPase activity, actin-sliding velocity, flight ability at room temperature or muscle structure are observed in IFI-9b compared to native IFI. However, when the EMB relay domain is replaced with the IFI relay domain, we find a 50% reduction in actin-activated ATPase activity, a significant increase in actin affinity, abolition of actin sliding, defects in myofibril assembly and rapid degeneration of muscle structure compared to EMB. We hypothesize that altered relay domain conformational changes in EMB-9a impair intramolecular communication with the EMB-specific converter domain. This decreases transition rates involving strongly bound actomyosin states, leading to a reduced ATPase rate and loss of actin motility.

Myosin transducer mutations differentially affect motor function, myofibril structure, and the performance of skeletal and cardiac muscles.

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc(5) affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc(5) (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc(5) myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc(5) mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.

Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster.

The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and/or stabilize thick filament interactions with proteins that contribute to fiber stiffness.