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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.

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 β-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 β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.

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.

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.

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.

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 β-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 β-cardiac myosin heavy chain cardiomyopathy mutations E497D or R712L, implying that abolishing this salt bridge is one cause of the human disease.

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 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.

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.

Hereditary inclusion-body myopathy type III (IBM-3) is caused by a single amino acid Glu706Lys substitution in the SH1 helix of the myosin head. The SH1 domain has been proposed to play a key role in the conformational changes that occur in the myosin head during force generation which is coupled to ATP hydrolysis. We are using an integrative approach to study the structure-function relationship of the myosin SH1 domain in the Drosophila model system. We constructed a gene encoding myosin with the single amino acid mutation and expressed it in place of wild-type myosin heavy chain by germline transformation and crossing into a line that lacks myosin in its flight and jump muscles. The homozygous flies are flightless and their jump abilities are also greatly reduced. The indirect flight muscle fibers of young flies show considerable ultrastructural disarray, with some regions of missing thick and thin filaments, and myofibrils that are not uniform in width. Our initial study showed that actin sliding velocity and basal and actin stimulated ATPase were reduced more than 70% compared to wild-type indirect flight muscle myosin. Homology models indicate that the surface charge change of the substitution in the highly conversed SH1 region could destabilize the helix, which is critical for the converter domain to rotate to its full range of movement during the power-stroke. This structural change would affect the lever arm swing, resulting in dysfunctional myosin. Given that human IBM-3 is mild in childhood but severe during aging, with the accumulation of inclusion bodies, we are investigating whether inclusion bodies or aggregates appear in aged mutant Drosophila muscle tissues.

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.