Shortening deactivation: quantifying a critical component of cyclical muscle contraction.

A muscle undergoing cyclical contractions requires fast and efficient muscle activation and relaxation to generate high power with relatively low energetic cost. To enhance activation and increase force levels during shortening, some muscle types have evolved stretch activation (SA), a delayed increased in force following rapid muscle lengthening. SA's complementary phenomenon is shortening deactivation (SD), a delayed decrease in force following muscle shortening. SD increases muscle relaxation, which decreases resistance to subsequent muscle lengthening. Although it might be just as important to cyclical power output, SD has received less investigation than SA. To enable mechanistic investigations into SD and quantitatively compare it to SA, we developed a protocol to elicit SA and SD from Drosophila and Lethocerus indirect flight muscles (IFM) and Drosophila jump muscle. When normalized to isometric tension, Drosophila IFM exhibited a 118% SD tension decrease, Lethocerus IFM dropped by 97%, and Drosophila jump muscle decreased by 37%. The same order was found for normalized SA tension: Drosophila IFM increased by 233%, Lethocerus IFM by 76%, and Drosophila jump muscle by only 11%. SD occurred slightly earlier than SA, relative to the respective length change, for both IFMs; but SD was exceedingly earlier than SA for jump muscle. Our results suggest SA and SD evolved to enable highly efficient IFM cyclical power generation and may be caused by the same mechanism. However, jump muscle SA and SD mechanisms are likely different, and may have evolved for a role other than to increase the power output of cyclical contractions.

Myosin dilated cardiomyopathy mutation S532P disrupts actomyosin interactions, leading to altered muscle kinetics, reduced locomotion, and cardiac dilation in Drosophila.

Dilated cardiomyopathy (DCM), a life-threatening disease characterized by pathological heart enlargement, can be caused by myosin mutations that reduce contractile function. To better define the mechanistic basis of this disease, we employed the powerful genetic and integrative approaches available in Drosophila melanogaster. To this end, we generated and analyzed the first fly model of human myosin-induced DCM. The model reproduces the S532P human ß-cardiac myosin heavy chain DCM mutation, which is located within an actin-binding region of the motor domain. In concordance with the mutation's location at the actomyosin interface, steady-state ATPase and muscle mechanics experiments revealed that the S532P mutation reduces the rates of actin-dependent ATPase activity and actin binding and increases the rate of actin detachment. The depressed function of this myosin form reduces the number of cross-bridges during active wing beating, the power output of indirect flight muscles, and flight ability. Further, S532P mutant hearts exhibit cardiac dilation that is mutant gene dose-dependent. Our study shows that Drosophila can faithfully model various aspects of human DCM phenotypes and suggests that impaired actomyosin interactions in S532P myosin induce contractile deficits that trigger the disease.

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.

Force-velocity and tension transient measurements from Drosophila jump muscle reveal the necessity of both weakly-bound cross-bridges and series elasticity in models of muscle contraction.

Muscle contraction is a fundamental biological process where molecular interactions between the myosin molecular motor and actin filaments result in contraction of a whole muscle, a process spanning size scales differing in eight orders of magnitude. Since unique behavior is observed at every scale in between these two extremes, to fully understand muscle function it is vital to develop multi-scale models. Based on simulations of classic measurements of muscle heat generation as a function of work, and shortening rate as a function of applied force, we hypothesize that a model based on molecular measurements must be modified to include a weakly-bound interaction between myosin and actin in order to fit measurements at the muscle fiber or whole muscle scales. This hypothesis is further supported by the model's need for a weakly-bound state in order to qualitatively reproduce the force response that occurs when a muscle fiber is rapidly stretched a small distance. We tested this hypothesis by measuring steady-state force as a function of shortening velocity, and the force transient caused by a rapid length step in Drosophila jump muscle fibers. Then, by performing global parameter optimization, we quantitatively compared the predictions of two mathematical models, one lacking a weakly-bound state and one with a weakly-bound state, to these measurements. Both models could reproduce our force-velocity measurements, but only the model with a weakly-bound state could reproduce our force transient measurements. However, neither model could concurrently fit both measurements. We find that only a model that includes weakly-bound cross-bridges with force-dependent detachment and an elastic element in series with the cross-bridges is able to fit both of our measurements. This result suggests that the force response after stretch is not a reflection of distinct steps in the cross-bridge cycle, but rather arises from the interaction of cross-bridges with a series elastic element. Additionally, the model suggests that the curvature of the force-velocity relationship arises from a combination of the force-dependence of weakly- and strongly-bound cross-bridges. Overall, this work presents a minimal cross-bridge model that has predictive power at the fiber level.

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.

A role for actin flexibility in thin filament-mediated contractile regulation and myopathy.

Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.

Enhancing diastolic function by strain-dependent detachment of cardiac myosin crossbridges.

The force response of cardiac muscle undergoing a quick stretch is conventionally interpreted to represent stretching of attached myosin crossbridges (phase 1) and detachment of these stretched crossbridges at an exponential rate (phase 2), followed by crossbridges reattaching in increased numbers due to an enhanced activation of the thin filament (phases 3 and 4). We propose that, at least in mammalian cardiac muscle, phase 2 instead represents an enhanced detachment rate of myosin crossbridges due to stretch, phase 3 represents the reattachment of those same crossbridges, and phase 4 is a passive-like viscoelastic response with power-law relaxation. To test this idea, we developed a two-state model of crossbridge attachment and detachment. Unitary force was assigned when a crossbridge was attached, and an elastic force was generated when an attached crossbridge was displaced. Attachment rate, f(x), was spatially distributed with a total magnitude f0. Detachment rate was modeled as g(x) = g0+ g1x, where g0 is a constant and g1 indicates sensitivity to displacement. The analytical solution suggested that the exponential decay rate of phase 2 represents (f0 + g0) and the exponential rise rate of phase 3 represents g0. The depth of the nadir between phases 2 and 3 is proportional to g1. We prepared skinned mouse myocardium and applied a 1% stretch under varying concentrations of inorganic phosphate (Pi). The resulting force responses fitted the analytical solution well. The interpretations of phases 2 and 3 were consistent with lower f0 and higher g0 with increasing Pi. This novel scheme of interpreting the force response to a quick stretch does not require enhanced thin-filament activation and suggests that the myosin detachment rate is sensitive to stretch. Furthermore, the enhanced detachment rate is likely not due to the typical detachment mechanism following MgATP binding, but rather before MgADP release, and may involve reversal of the myosin power stroke.

A myosin-based mechanism for stretch activation and its possible role revealed by varying phosphate concentration in fast and slow mouse skeletal muscle fibers.

Stretch activation (SA) is a delayed increase in force following a rapid muscle length increase. SA is best known for its role in asynchronous insect flight muscle, where it has replaced calcium's typical role of modulating muscle force levels during a contraction cycle. SA also occurs in mammalian skeletal muscle but has previously been thought to be too low in magnitude, relative to calcium-activated (CA) force, to be a significant contributor to force generation during locomotion. To test this supposition, we compared SA and CA force at different Pi concentrations (0-16 mM) in skinned mouse soleus (slow-twitch) and extensor digitorum longus (EDL; fast-twitch) muscle fibers. CA isometric force decreased similarly in both muscles with increasing Pi, as expected. SA force decreased with Pi in EDL (40%), leaving the SA to CA force ratio relatively constant across Pi concentrations (17-25%). In contrast, SA force increased in soleus (42%), causing a quadrupling of the SA to CA force ratio, from 11% at 0 mM Pi to 43% at 16 mM Pi, showing that SA is a significant force modulator in slow-twitch mammalian fibers. This modulation would be most prominent during prolonged muscle use, which increases Pi concentration and impairs calcium cycling. Based upon our previous Drosophila myosin isoform studies and this work, we propose that in slow-twitch fibers a rapid stretch in the presence of Pi reverses myosin's power stroke, enabling quick rebinding to actin and enhanced force production, while in fast-twitch fibers, stretch and Pi cause myosin to detach from actin.

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.

The load dependence of muscle's force-velocity curve is modulated by alternative myosin converter domains.

The hyperbolic shape of the muscle force-velocity relationship (FVR) is characteristic of all muscle fiber types. The degree of curvature of the hyperbola varies between muscle fiber types and is thought to be set by force-dependent properties of different myosin isoforms. However, the structural elements in myosin and the mechanism that determines force dependence are unresolved. We tested our hypothesis that the myosin converter domain plays a critical role in the force-velocity relationship (FVR) mechanism. Drosophila contains a single myosin heavy chain gene with five converters encoded by alternative exons. We measured FVR properties of Drosophila jump muscle fibers from five transgenic lines each expressing a single converter. Consistent with our hypothesis, we observed up to 2.4-fold alterations in FVR curvature. Maximum shortening velocity (v0) and optimal velocity for maximum power generation were also altered, but isometric tension and maximum power generation were unaltered. Converter 11a, normally found in the indirect flight muscle (IFM), imparted the highest FVR curvature and v0, whereas converter 11d, found in larval body wall muscle, imparted the most linear FVR and slowest v0. Jump distance strongly correlated with increasing FVR curvature and v0, meaning flies expressing the converter from the IFM jumped farther than flies expressing the native jump muscle converter. Fitting our data with Huxley's two-state model and a biophysically based four-state model suggest a testable hypothesis that the converter sets muscle type FVR curvature by influencing the detachment rate of negatively strained myosin via changes in the force dependence of product release.

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.

Five Alternative Myosin Converter Domains Influence Muscle Power, Stretch Activation, and Kinetics.

Muscles have evolved to power a wide variety of movements. A protein component critical to varying power generation is the myosin isoform present in the muscle. However, how functional variation in muscle arises from myosin structure is not well understood. We studied the influence of the converter, a myosin structural region at the junction of the lever arm and catalytic domain, using Drosophila because its single myosin heavy chain gene expresses five alternative converter versions (11a-e). We created five transgenic fly lines, each forced to express one of the converter versions in their indirect flight muscle (IFM) fibers. Electron microscopy showed that the converter exchanges did not alter muscle ultrastructure. The four lines expressing converter versions (11b-e) other than the native IFM 11a converter displayed decreased flight ability. IFM fibers expressing converters normally found in the adult stage muscles generated up to 2.8-fold more power and displayed up to 2.2-fold faster muscle kinetics than fibers with converters found in the embryonic and larval stage muscles. Small changes to stretch-activated force generation only played a minor role in altering power output of IFM. Muscle apparent rate constants, derived from sinusoidal analysis of the chimeric converter fibers, showed a strong positive correlation between optimal muscle oscillation frequency and myosin attachment kinetics to actin, and an inverse correlation with detachment related cross-bridge kinetics. This suggests the myosin converter alters at least two rate constants of the cross-bridge cycle with changes to attachment and power stroke related kinetics having the most influence on setting muscle oscillatory power kinetics.

Stretch activation properties of Drosophila and Lethocerus indirect flight muscle suggest similar calcium-dependent mechanisms.

Muscle stretch activation (SA) is critical for optimal cardiac and insect indirect flight muscle (IFM) power generation. The SA mechanism has been investigated for decades with many theories proposed, but none proven. One reason for the slow progress could be that multiple SA mechanisms may have evolved in multiple species or muscle types. Laboratories studying IFM SA in the same or different species have reported differing SA functional properties which would, if true, suggest divergent mechanisms. However, these conflicting results might be due to different experimental methodologies. Thus, we directly compared SA characteristics of IFMs from two SA model systems, Drosophila and Lethocerus, using two different fiber bathing solutions. Compared with Drosophila IFM, Lethocerus IFM isometric tension is 10- or 17-fold higher and SA tension was 5- or 10-fold higher, depending on the bathing solution. However, the rate of SA tension generation was 9-fold faster for Drosophila IFM. The inverse differences between rate and tension in the two species causes maximum power output to be similar, where Drosophila power is optimized in the bathing solution that favors faster muscle kinetics and Lethocerus in the solution that favors greater tension generation. We found that isometric tension and SA tension increased with calcium concentration for both species in both solutions, reaching a maximum plateau around pCa 5.0. Our results favor a similar mechanism for both species, perhaps involving a troponin complex that does not fully calcium activate the thin filament thus leaving room for further tension generation by SA.

A Drosophila model of dominant inclusion body myopathy type 3 shows diminished myosin kinetics that reduce muscle power and yield myofibrillar defects.

Individuals with inclusion body myopathy type 3 (IBM3) display congenital joint contractures with early-onset muscle weakness that becomes more severe in adulthood. The disease arises from an autosomal dominant point mutation causing an E706K substitution in myosin heavy chain type IIa. We have previously expressed the corresponding myosin mutation (E701K) in homozygous Drosophila indirect flight muscles and recapitulated the myofibrillar degeneration and inclusion bodies observed in the human disease. We have also found that purified E701K myosin has dramatically reduced actin-sliding velocity and ATPase levels. Since IBM3 is a dominant condition, we now examine the disease state in heterozygote Drosophila in order to gain a mechanistic understanding of E701K pathogenicity. Myosin ATPase activities in heterozygotes suggest that approximately equimolar levels of myosin accumulate from each allele. In vitro actin sliding velocity rates for myosin isolated from the heterozygotes were lower than the control, but higher than for the pure mutant isoform. Although sarcomeric ultrastructure was nearly wild type in young adults, mechanical analysis of skinned indirect flight muscle fibers revealed a 59% decrease in maximum oscillatory power generation and an approximately 20% reduction in the frequency at which maximum power was produced. Rate constant analyses suggest a decrease in the rate of myosin attachment to actin, with myosin spending decreased time in the strongly bound state. These mechanical alterations result in a one-third decrease in wing beat frequency and marginal flight ability. With aging, muscle ultrastructure and function progressively declined. Aged myofibrils showed Z-line streaming, consistent with the human heterozygote phenotype. Based upon the mechanical studies, we hypothesize that the mutation decreases the probability of the power stroke occurring and/or alters the degree of movement of the myosin lever arm, resulting in decreased in vitro motility, reduced muscle power output and focal myofibrillar disorganization similar to that seen in individuals with IBM3.

The Drosophila indirect flight muscle myosin heavy chain isoform is insufficient to transform the jump muscle into a highly stretch-activated muscle type.

Stretch activation (SA) is a delayed increase in force that enables high power and efficiency from a cyclically contracting muscle. SA exists in various degrees in almost all muscle types. In Drosophila, the indirect flight muscle (IFM) displays exceptionally high SA force production (FSA), whereas the jump muscle produces only minimal FSA We previously found that expressing an embryonic (EMB) myosin heavy chain (MHC) isoform in the jump muscle transforms it into a moderately SA muscle type and enables positive cyclical power generation. To investigate whether variation in MHC isoforms is sufficient to produce even higher FSA, we substituted the IFM MHC isoform (IFI) into the jump muscle. Surprisingly, we found that IFI only caused a 1.7-fold increase in FSA, less than half the increase previously observed with EMB, and only at a high Pi concentration, 16 mM. This IFI-induced FSA is much less than what occurs in IFM, relative to isometric tension, and did not enable positive cyclical power generation by the jump muscle. Both isometric tension and FSA of control fibers decreased with increasing Pi concentration. However, for IFI-expressing fibers, only isometric tension decreased. The rate of FSA generation was ~1.5-fold faster for IFI fibers than control fibers, and both rates were Pi dependent. We conclude that MHC isoforms can alter FSA and hence cyclical power generation but that isoforms can only endow a muscle type with moderate FSA Highly SA muscle types, such as IFM, likely use a different or additional mechanism.

A Restrictive Cardiomyopathy Mutation in an Invariant Proline at the Myosin Head/Rod Junction Enhances Head Flexibility and Function, Yielding Muscle Defects in Drosophila.

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 new experimental model for force enhancement: steady-state and transient observations of the Drosophila jump muscle.

The increase in steady-state force after active lengthening in skeletal muscle, termed force enhancement (FE), has been observed for nearly one century. Although demonstrated experimentally at various structural levels, the underlying mechanism(s) remain unknown. We recently showed that the Drosophila jump muscle is an ideal model for investigating mechanisms behind muscle physiological properties, because its mechanical characteristics, tested thus far, duplicate those of fast mammalian skeletal muscles, and Drosophila has the advantage that it can be more easily genetically modified. To determine if Drosophila would be appropriate to investigate FE, we performed classic FE experiments on this muscle. Steady-state FE (FESS), following active lengthening, increased by 3, 7, and 12% of maximum isometric force, with increasing stretch amplitudes of 5, 10, and 20% of optimal fiber length (FLOPT), yet was similar for stretches across increasing stretch velocities of 4, 20, and 200% FLOPT/s. These FESS characteristics of the Drosophila jump muscle closely mimic those observed previously. Jump muscles also displayed typical transient FE characteristics. The transient force relaxation following active stretch was fit with a double exponential, yielding two phases of force relaxation: a fast initial relaxation of force, followed by a slower recovery toward steady state. Our analyses identified a negative correlation between the slow relaxation rate and FESS, indicating that there is likely an active component contributing to FE, in addition to a passive component. Herein, we have established the Drosophila jump muscle as a new and genetically powerful experimental model to investigate the underlying mechanism(s) of FE.

The roles of troponin C isoforms in the mechanical function of Drosophila indirect flight muscle.

Stretch activation (SA) is a fundamental property of all muscle types that increases power output and efficiency, yet its mechanism is unknown. Recently, studies have implicated troponin isoforms as important in the SA mechanism. The highly stretch-activated Drosophila IFMs express two isoforms of the Ca(2+)-binding subunit of troponin (TnC). TnC1 (TnC-F2 in Lethocerus IFM) has two calcium binding sites, while an unusual isoform, TnC4 (TnC-F1 in Lethocerus IFM), has only one binding site. We investigated the roles of these two TnC isoforms in Drosophila IFM by targeting RNAi to each isoform. IFMs with TnC4 expression (normally ~90% of total TnC) replaced by TnC1 did not generate isometric tension, power or display SA. However, TnC4 knockdown resulted in sarcomere ultrastructure disarray, which could explain the lack of mechanical function and thus make interpretation of the influence of TnC4 on SA difficult. Elimination of TnC1 expression (normally ~10% of total TnC) by RNAi resulted in normal muscle structure. In these IFMs, fiber power generation, isometric tension, stretch-activated force and calcium sensitivity were statistically identical to wild type. When TnC1 RNAi was driven by an IFM specific driver, there was no decrease in flight ability or wing beat frequency, which supports our mechanical findings suggesting that TnC1 is not essential for the mechanical function of Drosophila IFM. This finding contrasts with previous work in Lethocerus IFM showing TnC1 is essential for maximum isometric force generation. We propose that differences in TnC1 function in Lethocerus and Drosophila contribute to the ~40-fold difference in IFM isometric tension generated between these species.

A new experimental model to study force depression: the Drosophila jump muscle.

Force depression (FD) is a decrease in isometric force following active muscle shortening. Despite being well characterized experimentally, its underlying mechanism remains unknown. To develop a new, genetically manipulatable experimental model that would greatly improve our ability to study the underlying mechanism(s) of FD, we tested the Drosophila jump muscle for classical FD behavior. Steady-state force generation following active shortening decreased by 2, 8, and 11% of maximum isometric force with increasing shortening amplitudes of 5, 10, and 20% of optimal fiber length, and decreased by 11, 8, and 5% with increasing shortening velocities of 4, 20, and 200% of optimal fiber length per second. These steady-state FD (FDSS) characteristics of Drosophila jump muscle mimic those observed in mammalian skeletal muscle. A double exponential fit of transient force recovery following shortening identified two separate phases of force recovery: a rapid initial force redevelopment, and a slower recovery toward steady state. This analysis showed the slower rate of force redevelopment to be inversely proportional to the amount of FDSS, while the faster rate did not correlate with FDSS. This suggests that the mechanism behind the slower, most likely cross-bridge cycling rate, influences the amount of FDSS. Thus the jump muscle, when coupled with the genetic mutability of its sarcomere proteins, offers a unique and powerful experimental model to explore the underlying mechanism behind FD.

An embryonic myosin converter domain influences Drosophila indirect flight muscle stretch activation, power generation and flight.

Stretch activation (SA) is critical to the flight ability of insects powered by asynchronous, indirect flight muscles (IFMs). An essential muscle protein component for SA and power generation is myosin. Which structural domains of myosin are significant for setting SA properties and power generation levels is poorly understood. We made use of the transgenic techniques and unique single muscle myosin heavy chain gene of Drosophila to test the influence of the myosin converter domain on IFM SA and power generation. Replacing the endogenous converter with an embryonic version decreased SA tension and the rate of SA tension generation. The alterations in SA properties and myosin kinetics from the converter exchange caused power generation to drop to 10% of control fiber power when the optimal conditions for control fibers - 1% muscle length (ML) amplitude and 150 Hz oscillation frequency - were applied to fibers expressing the embryonic converter (IFI-EC). Optimizing conditions for IFI-EC fiber power production, by doubling ML amplitude and decreasing oscillation frequency by 60%, improved power output to 60% of optimized control fiber power. IFI-EC flies altered their aerodynamic flight characteristics to better match optimal fiber power generation conditions as wing beat frequency decreased and wing stroke amplitude increased. This enabled flight in spite of the drastic changes to fiber mechanical performance.