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K146N is a dominant mutation in human β-cardiac myosin heavy chain, which causes hypertrophic cardiomyopathy. We examined howDrosophilamuscle 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 mutatedDrosophilahearts could rapidly identify hypertrophic cardiomyopathy alleles and treatments.

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.

An "invariant proline" separates the myosin S1 head from its S2 tail and is proposed to be critical for orienting S1 during its interaction with actin, a process that leads to muscle contraction. Mutation of the invariant proline to leucine (P838L) caused dominant restrictive cardiomyopathy in a pediatric patient (Karam et al., Congenit. Heart Dis. 3:138-43, 2008). Here, we use Drosophila melanogaster to model this mutation and dissect its effects on the biochemical and biophysical properties of myosin, as well as on the structure and physiology of skeletal and cardiac muscles. P838L mutant myosin isolated from indirect flight muscles of transgenic Drosophila showed elevated ATPase and actin sliding velocity in vitro. Furthermore, the mutant heads exhibited increased rotational flexibility, and there was an increase in the average angle between the two heads. Indirect flight muscle myofibril assembly was minimally affected in mutant homozygotes, and isolated fibers displayed normal mechanical properties. However, myofibrils degraded during aging, correlating with reduced flight abilities. In contrast, hearts from homozygotes and heterozygotes showed normal morphology, myofibrillar arrays, and contractile parameters. When P838L was placed in trans to Mhc(5), an allele known to cause cardiac restriction in flies, it did not yield the constricted phenotype. Overall, our studies suggest that increased rotational flexibility of myosin S1 enhances myosin ATPase and actin sliding. Moreover, instability of P838L myofibrils leads to decreased function during aging of Drosophila skeletal muscle, but not cardiac muscle, despite the strong evolutionary conservation of the P838 residue.

A Drosophila model of myosin-based inclusion body myopathy type 3 is presented. Muscle function, ATPase activity, and actin sliding velocity were dramatically reduced. The mutant myosin is prone to aggregate, likely accounting for the observed cytoplasmic inclusions and disorganized muscle filaments reminiscent of the human disease., 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.

Inclusion body myopathy type 3 (IBM-3) is a progressive dominant disease affecting fast skeletal muscle. It results from a point mutation in the SH1 helix of the myosin motor. Previously, we showed that homozygous expression of the analogous mutation in Drosophila indirect flight muscle (IFM) results in a flightless phenotype, severe abnormalities in myofibril structure, dramatically reduced ATPase and in vitro motility, increased myosin aggregation and production of autophagic and membranous inclusions (Wang et al., 2012, Mol Biol Cell 23:2057-65). We have now examined the dominant effects of the IBM-3 mutation in our model. Mutant and wild-type proteins accumulate in equimolar amounts in heterozygotes, since ATPase and in vitro motility levels display intermediate values. Heterozygotes show progressive defects in IFM function that are less severe than in homozygotes, correlating with deficits in fiber mechanics (Corcione et al., 2010, Biophys. J. 98: 544a). Surprisingly, we detect no defects in muscle morphology in heterozygotes. While increasing the mutant:wild-type gene dosage to 2:1 further compromises muscle function and produces minor myofibrillar defects in aged flies, no inclusion bodies are observed. We examined the accumulation of Ref(2)P, a polyubiquitin-binding protein that is commonly associated with protein aggregates, and found a dramatic upregulation only in the IBM-3 homozygotes. We defined the makeup of these aggregates by proteomic analysis and found alterations in proteins associated with mitochondrial function and the unfolded protein response. We plan to manipulate expression levels of such proteins in an attempt to improve mutant phenotypes.Supported by MDA grant 217900. We appreciate the assistance of Robert N. Cole and Robert O’Meally, JHU Mass Spectrometry and Proteomics Facility.

A human K146N hypertrophic cardiomyopathy (HCM) myosin mutation localizes near the N-terminus of β-cardiac MHC, and molecular modeling of the analogous Drosophila R146 residue predicts its electrostatic interaction with an E774 residue of the lever arm during the pre-power stroke state. We expressed the R146N mutation in Drosophila to test whether disrupting this interaction alters myosin structure and function and causes HCM. R146N mutants exhibited defects in flight ability and progressive disruptions in indirect flight muscle (IFM) morphology.

Interaction of the myosin relay domain with the converter domain and with the SH1-SH2 helix region appears to be vital for the power stroke and muscle contraction. To define involvement of specific residues in these interactions we used mutation and compensatory mutation approach. The mutant myosins were expressed transgenically in Drosophila indirect flight muscles, which allow assessment of myosin function in vitro and in vivo. We mutated residue R714 near the SH1-SH2 helix in the main body of myosin. We found that R714E myosin ATPase is reduced by ∼80% and that no in vitro motility is detected. R714E organisms show myofibril assembly defects that are exacerbated with age and these organisms cannot fly. Putative compensatory mutation E499R in the relay domain improves ATPase activity so that there is only ∼35% reduction compared to wild type. Further, R714E/E499R myosin moves actin filaments at ∼30% of wild-type velocity. Normal myofibril assembly is restored and myofibrils are more stable. We next studied mutation R759E in the converter domain. It depresses myosin ATPase activity, in vitro motility and flight ability. Although myofibrils assemble normally, they degenerate as flies age. We inserted a lysine mutation at putative interacting relay domain loop residue N509 in an attempt to suppress the R759E mutant phenotypes. N509K dramatically rescues muscle structure and function, with significant increases in ATPase rates and in vitro motility. These interactions are critical for normal ATPase activity, in vitro motility, myofibril assembly and/or stability and flight muscle function. Thus the specific residue contacts identified permit the myosin relay domain to communicate information between the nucleotide binding pocket and the converter domain and are essential for progress through the mechanochemical cycle that powers muscle contraction.

Hereditary myosin myopathies are muscle diseases with variable clinical features and age of onset. Inclusion body myopathy 3 (IBM3) is an autosomal dominant myopathy associated with a missense mutation (E706K) in the myosin heavy chain IIa gene (MYH2). The disease is mild in childhood but appears progressive in adulthood, with proximal muscle weakness affecting movement. Biopsies from adult patients reveal dystrophic alterations and rimmed vacuoles consistent with an increased expression of the mutant motor with advanced age. We constructed a transgene encoding E706K myosin and expressed it in Drosophila (E699K) indirect flight and jump muscles. Flight and jump abilities were reduced in heterozygous flies and were nearly absent in homozygotes consistent with the possible age-related dose-dependent response observed in patients. The mutant myosin displayed 80% lower actin sliding velocity and 74 and 83% reductions in basal and actin stimulated ATPase activities compared to wild-type myosin. Electron microscopy revealed E706K (E699K) myosin heads bear a dramatic propensity to collapse and to aggregate relative to wildtype heads. At 23°C, 77.5% (n=1146) of control molecules exhibited two well-resolved independent heads compared to only 22.5% (n=1014) of mutant myosin molecules. A five minute, 37°C incubation induced 80.9% (n=1181) of the control molecules’ heads to form intra- or intermolecular aggregates versus 95.3% (n=1192) of the mutant myosin heads. This test directly assessed motor integrity and suggests E706K (E699K) myosin is far more labile than wildtype myosin. We are imaging mutant myocytes to determine if the ultrastructural hallmarks seen in adult patients also appear in our fly model. The depressed motor properties and the propensity for the mutant myosin to collapse and aggregate likely contribute to the muscle weakness observed in our fly model and possibly in senescent patients.

We used an integrative approach to probe the significance of the interaction between the relay loop and converter domain of Drosophila melanogaster skeletal muscle myosin. We generated a transgenic line expressing myosin with a mutation in the converter domain (R759E) at the relay loop interaction site. The mutation depresses calcium, basal or actin-activated MgATPase values (Vmax) by ∼60% and actin sliding velocity ∼35% compared to wild-type myosin. Ultrastructure of two-day-old adult fibers shows cracking and frayed myofibrils with some disruption of the myofilament lattice which becomes more severe in one-week-old adults. Flight ability is reduced in two-day-old flies compared to controls and is absent in 1-week-old adults. Thus appropriate interaction between the relay loop and converter domain is essential for normal motor function, myofibril stability and locomotion. To examine the specificity of this interaction, we used a compensatory mutational approach to attempt to restore the function of the R759E mutant myosin. Our modeling indicates that relay loop residues N509 and D511 interact with converter domain residue R759. To verify our model, we generated two transgenic lines that express R759E and either the N509K or D511K mutations. Interestingly, calcium, basal, and actin stimulated ATPase values are restored to 70% and actin sliding velocity is restored to 90% in N509K/R759E but not in D511K/R759E. Structurally fibers from 2-day or one-week -old adults appear morphologically normal in N509K/R759E and their flight ability is like wild type. However, D511K/R759E myofibrils do not show any improvement compared to R759E and flight ability is worse than R759E. Overall, our results reveal the critical interaction between the converter domain with relay loop residues and their role in myosin motor function and myofibril assembly/stability.

Numerous myosin-based myopathies have been identified without a detailed understanding of their molecular basis. To pursue mechanistic investigations, we generated transgenic Drosophila expressing the R146N or R249Q myosin mutations that cause hypertrophic cardiomyopathy in humans. Our studies show severely compromised flight ability in homozygotes, with an age-dependent worsening of muscle function and myofibrillar disarray in the indirect flight muscle ultrastructure. The R146N mutation caused an increase in basal Ca-ATPase and Mg-ATPase activities, with a significant decrease in actin motility. Semi-automated optical heartbeat analysis performed using high-speed movies of semi-intact mutant heterozygous hearts indicated restrictive cardiac physiology and diastolic dysfunction. Based upon molecular modeling, we predict 1) the R146N mutation alters ionic interaction of the N-terminal motor domain with E774 of the lever arm, resulting in failure of the lever arm to cock prior to the power-stroke; and 2) the R249Q mutation disrupts electrostatic interaction with D262 of the central β-sheet domain, interrupting communication between the ATP-binding and actin-binding sites. Thus, the models imply that these conserved charge interactions are critical for myosin function. To test these predictions, lines expressing R146E, E774R, R249D and D262R mutations, as well as putative suppressor lines R146E+E774R and R249D+D262R, will be studied using an integrative biochemical, biophysical and physiological approach. Transgenic Drosophila expressing either R146E or R249D myosin revealed more severe phenotypes relative to cardiomyopathy mutations. Both homozygous mutants displayed flightless phenotypes and highly compromised myofibrillar ultrastructure, with the R146E mutants showing increased basal Ca-ATPase and Mg-ATPase activities, and no actin motility. Heterozygotes present with severe restricted cardiac physiology and diastolic dysfunction. Overall our studies indicate that the Drosophila is a valuable tool to test significant myosin interactions during health and disease. Supported by NIH R01GM32443 to SIB.