Mutations that affect flightin expression in Drosophila alter the viscoelastic properties of flight muscle fibers

Josh A. Henkin,1,2,3 David W. Maughan,2,3 and Jim O. Vigoreaux1,2,3

1Department of Biology, 2Department of Molecular Physiology and Biophysics, and 3Cell and Molecular Biology Program, University of Vermont, Burlington, Vermont 05405

Submitted 20 June 2003 ; accepted in final form 31 August 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Striated muscles across phyla share a highly conserved sarcomere design yet exhibit broad diversity in contractile velocity, force, power output, and efficiency. Insect asynchronous flight muscles are characterized by high-frequency contraction, endurance, and high-power output. These muscles have evolved an enhanced delayed force response to stretch that is largely responsible for their enhanced oscillatory work and power production. In this study we investigated the contribution of flightin to oscillatory work using sinusoidal analysis of fibers from three flightless mutants affecting flightin expression: 1) fln0, a flightin null mutant, 2) Mhc13, a myosin rod point mutant with reduced levels of flightin, and 3) Mhc6, a second myosin rod point mutant with reduced levels of phosphorylated flightin. Fibers from the three mutants show deficits in their passive and dynamic viscoelastic properties that are commensurate with their effect on flightin expression and result in a significant loss of oscillatory work and power. Passive tension and passive stiffness were significantly reduced in fln0 and Mhc13 but not in Mhc6. The dynamic viscous modulus was significantly reduced in the three mutants, whereas the dynamic elastic modulus was reduced in fln0 and Mhc13 but not in Mhc6. Tension generation under isometric conditions was not impaired in fln0. However, when subjected to sinusoidal length perturbations, work-absorbing processes dominated over work-producing processes, resulting in no net positive work output. We propose that flightin is a major contributor to myofilament stiffness and a key determinant of the enhanced delayed force response to stretch in Drosophila flight muscles.

flight muscles; muscle mutants; myosin


THERE ARE MORE SPECIES OF INSECTS than of any other type of animal. Although the exceptional diversity in insect species is the result of many factors, differences in flight ability are generally considered among the most important ones. Insects were the first organisms to take to the air approximately 450 million years ago. Because flight is not only a means of locomotion but is also used for mating, prey capture, and defense, multiple factors have influenced the evolution of flight systems, giving rise to varied and extraordinary performers. The study of insect flight muscle thus promises to uncover evolutionarily conserved features as well as specialized adaptations in muscle function.

Insect flight is powered by striated muscles situated within the thorax. In particular, the asynchronous indirect flight muscles (IFM), found in dipterans and several other orders, are generally considered among the most powerful muscles in the animal kingdom (9, 10). In these muscles, a delayed increase in tension following a stretch (the so-called stretch activation response) is very prominent and underlies the ability of the muscle to produce work against a load (24). IFM are also characterized by a high resting stiffness, an important feature given that passive muscle properties are generally believed to influence the mechanical performance of active muscle (36). Passive stiffness is thought to reside largely within the connecting filaments, structures that connect the ends of thick filaments to the Z band (8). These filaments consist of projectin, and possibly kettin, proteins that belong to the immunoglobulin superfamily of gigantic proteins (2, 12, 27). In vertebrate striated muscles, titin has been shown to be responsible for generating passive forces when sarcomeres are stretched above or shortened below their slack length (14). Furthermore, the diversity in elastic properties among vertebrate striated muscles is largely explained by differences in titin isoforms generated by alternative splicing (35). Projectin (0.9 MDa) and kettin (0.5–0.8 MDa) are much shorter than titin (2.0–3.0 MDa), and their presence in IFM likely accounts for the higher resting stiffness of this muscle compared with vertebrate skeletal and cardiac muscle (12).

Another characteristic feature of insect IFM is the presence of ancillary myofibrillar proteins that have no homologous counterparts in vertebrates (31). One such protein is flightin, a 20,000-dalton myosin rod binding protein with multiple phosphorylation sites (1, 26, 33). In Drosophila, flightin is expressed in the IFM exclusively, and a null mutation in the flightin gene (fln0) has no effect on viability. Nevertheless, fln0 flies are flightless because their IFM undergo a time-dependent degeneration beginning shortly after eclosion (the pupal-to-adult transition) (26). Flightin's essentiality for thick filament stability in actively contracting muscle is also evident in Mhc13, a point mutation in the myosin heavy chain (MHC) rod that prevents normal flightin accumulation. In this mutant, the IFM undergoes a time-dependent degeneration that mirrors the degeneration seen in fln0 (11). The sarcomere breakdown in Mhc13 and fln0 is accompanied by, or results from, proteolytic cleavage near the S2 hinge region of myosin, suggesting that the presence of flightin is essential for preserving myosin integrity in active muscle (11, 26). Flightin also plays an important role in thick filament length determination as evidenced by the presence of longer than normal thick filaments and sarcomeres in fln0 pupal IFM (26).

We conducted the present study to examine the mechanical properties of mutant Drosophila IFM with altered flightin expression. We examined three mutants in which the sarcomere structure is well preserved in very young adults but in which expression of flightin is affected to different degrees: 1) fln0 lacks flightin completely; 2) Mhc13 prevents the accumulation of all flightin isovariants except for reduced amounts of the nonphosphorylated form (N1); and 3) Mhc6, another myosin rod single amino acid mutation, prevents complete phosphorylation of flightin but permits accumulation of nonphosphorylated flightin and a limited subset of its phosphovariants (11).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Fly stocks. fln0 is a recessive flightless Drosophila strain with a null mutation in the flightin gene (26). The dominant flightless MHC mutant strains Mhc6 and Mhc13 were obtained from Sanford Bernstein (San Diego State University, San Diego, CA) and are described by Kronert et al. (11). Both mutations are single amino acid substitutions in the light meromyosin (LMM) region of MHC. None of the mutant strains were able to beat their wings. For controls, we used the wild-type Canton S strain.

Skinned fiber mechanics. The mechanical properties of skinned dorsal longitudinal muscle fibers were assessed as previously described (4). Female flies <2 h old (most within 30 min of eclosion) were chosen to avoid time-dependent degradation of the IFM [cellular damage (tearing or bunching)] otherwise evident under the dissecting microscope in all three mutants. Only fibers that did not exhibit morphological defects at the time of dissection were used. Although microscopic inspection cannot detect subcellular damage, if present, previous studies have shown that in newly eclosed mutant flies, sarcomere structure is preserved and is virtually indistinguishable from that of newly eclosed wild-type flies (11, 26).

All reagents were purchased from Sigma (St. Louis, MO). Fibers were dissected in a York modified glycerol solution (20 mM KPi buffer, 4 mM DTT, 2 mM MgCl2, 0.5% Triton, 50% glycerol) containing 5 µg/l Sigma cocktail protease inhibitor (26). Fibers were skinned in YMG for 1 h at 4°C, after which aluminum T clips were affixed to each end of a single muscle fiber (23). One clip was attached to a piezoelectric motor, and the second was attached to a force transducer. The fiber was then lowered into a 15°C, 30-µl drop of relaxing solution [5 mM ATP, 15 mM creatine phosphate, 240 U/ml creatine phosphokinase, 1 mM free Mg2+, 5 mM EGTA, and 20 mM N,N-bis(2-hydroxyethly)-2-aminoethanesulfonic acid (BES) (pH 7.0), at an ionic strength of 175 mM adjusted with sodium methanesulfonate] with 0.5% Triton X-100 (pCa 8.0) and positioned above a glass-bottomed chamber surrounded by 1 ml of mineral oil at 15°C.

Before Ca2+ activation or sinusoidal perturbation, fibers were prestretched by increments of 2% of their initial length and monitored for changes in tension. The stretch was terminated when fiber tension increased to 1 kN/m2 from baseline or when the stretch reached 10% of the fiber's initial length, whichever came first. Fibers were activated progressively by exchanging equal volumes of activating solution (pCa 4.5) for relaxing solution (pCa 8.0) up to pCa 5.0. Exchanged values were determined by using custom software to take into account the various ionic species present in the solution (6). Isometric tension was recorded at each pCa. Sinusoidal length changes were applied over 47 frequencies from 0.5 to 1,000 Hz. At each frequency, the length and tension signals were measured to determine the fiber's complex stiffness modulus [Y(f)]. The complex stiffness modulus is defined as the ratio of stress to strain in the frequency domain, where stress is the force (F) per cross-sectional area (A) and strain is the fractional length change ({Delta}L; L0 = initial length): Y(f) = ({Delta}F/A)/({Delta}L/L0). The frequency-response function of a latex strip was used to correct for system characteristics.

Upon reaching pCa 5.0, the activating solution was replaced entirely with a rigor solution [5 mM EGTA and 20 mM BES (pH 7.0), at an ionic strength of 175 mM adjusted with sodium methanesulfonate]. Sinusoidal length changes were applied once again over the same frequencies, and complex stiffness modulus was obtained for one rigor cycle. A large percentage of mutant fibers ripped or broke apart during the rigor cycle. In addition, we conducted an abbreviated mechanical protocol during which Ca2+-activated fibers were placed in rigor without undergoing sinusoidal length changes. Two fibers from each strain were tested in this manner to establish whether the mechanical regime exacerbated fiber damage observed in rigor. Fiber rundown was estimated from the attenuation of maximum work output (proportional to the amplitude of maximum negative viscous modulus, –Ev), i.e., the ratio of –Ev measured at pCa 5.0 at the end of the experiment divided by –Ev measured at pCa 5.0 at the beginning of the experiment. Based on data from parallel experiments on wild-type and mutant lines, fiber rundown was 0.93 ± 0.04 (mean ± SE), i.e., nominally, an ~7% decrement of work-producing capacity over the course of the experiment.

Cross bridge stiffness. To determine the fractional stiffness due to cross bridges, the relaxed stiffness (pCa 8.0 at 1.0 Hz) was subtracted from the active stiffness (pCa 5.0 at 1.0 Hz) and the result was divided the by the active stiffness. It was not possible to measure rigor stiffness because most mutant fibers collapsed or broke apart upon removal of ATP.

Statistical analysis. Data were analyzed using SPSS version 11.0.1 (SPSS, Chicago, IL). The Kruskal-Wallis nonparametric test was performed to test for differences in means among the groups, because data violated Levene's test for equal variance among groups. If overall significance was found, the Mann-Whitney post hoc test was used for pairwise comparisons to identify which group means differed from one another. Hill fit coefficients were compared by using the procedure for testing the difference between two regression coefficients. An alpha level of 0.05 was required for significance.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Distinct effects of mutations on isometric tension and Ca2+ sensitivity. Ca2+-dependent isometric tension was measured over the pCa range from 8.0 to 5.0. The average tension for each of the three mutants over this range was lower compared with wild-type controls. However, except for Mhc13, the differences were not statistically significant (Table 1). Tension for Mhc13 was approximately one-third the tension in wild type (P < 0.005). The two myosin mutations caused an increase in the Ca2+ sensitivity of the fiber (pCa50 of 6.11 and 6.42, respectively, for Mhc6 and Mhc13, vs. 5.59 for wild type; Table 1). Both myosin mutations caused a decrease in the Hill coefficient, an index of the cooperativeness of Ca2+ activation. Fibers from fln0 are essentially insensitive to low [Ca2+], producing force only at pCa 5.5 in a stepwise activation response. For this reason, we were not able to calculate pCa50 or Hill coefficient for this mutant (Table 1). The stepwise response of fln0 fibers to Ca2+ may be related to the longer than normal sarcomeres that are typical of this mutant in newly eclosed adults (26). The ability of the mutant fibers to produce Ca2+-dependent tension suggests that their primary effects are not on the force-generating properties of the myosin motor.


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Table 1. Isometric tension, passive stiffness, and Ca2+ sensitivity of normal and mutant skinned fibers

 

Reduced passive tension and stiffness in mutant fibers. Fibers from young fln0 adults are abnormally wavy (26). Before their mechanical properties were measured, skinned fibers mounted on the apparatus were stretched taut to a level of resting stress equivalent to ~1 kN/m2. The average prestretch value for wild type and Mhc6 is 4 and 5% of their original length, respectively, whereas for Mhc13 and fln0 the stretch value is approximately two times greater (8%). This difference may arise from greater compliance of elements within each sarcomere, longer sarcomeres, which may be responsible for the abnormal waviness, or both.

Mutant fibers showed reductions in passive tension for a given stretch that were commensurate with their levels of flightin accumulation (Fig. 1). Fibers from Mhc6 exhibited tension levels that were not significantly different from wild type. In contrast, fibers from Mhc13 and fln0 showed tension values that were significantly reduced compared with wild type. A steep initial increase in tension upon stretch that continued to rise at higher strains was observed in wild-type and Mhc6 fibers. In contrast, Mhc13 and fln0 exhibited a plateau at short lengths (2–4% stretch) but, similar to wild type, reached higher levels of tension at longer lengths. The pronounced plateau seen in fln0 and Mhc13 suggests that some myofibrillar element(s), perhaps flightin, contributes to passive tension over the limited range of length amplitudes through which the muscle operates in vivo [2–5%, (3)]. Hysteresis, evident as lower tension during the release compared with that during stretch, was present in all fibers.



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Fig. 1. Passive tension-length relations of wild-type and mutant skinned indirect flight muscle (IFM) fibers. Fibers were stretched to 10% of their resting length and then released in increments of 2%. Each curve represents the average of 3 fibers (mean ± SE). The increase in passive tension with stretch is very pronounced for wild-type and Mhc6 fibers but much reduced for Mhc13 and fln0 fibers.

 

Static (chord) stiffness measured in the relaxed fiber (pCa 8.0) as the ratio of stress to strain also revealed differences among the mutants (Table 1). Both fln0 and Mhc13 had considerably lower stiffness compared with wild type, whereas Mhc6 had no effect on passive stiffness.

Altered dynamic properties in mutant fibers. Sinusoidal analysis was used to measure the viscoelastic properties of maximally Ca2+-activated fibers. All three mutations resulted in decreased dynamic stiffness. The magnitude of these changes parallels the decrease in passive stiffness, with Mhc6 showing a nonsignificant decrease and Mhc13 showing a highly significant decrease (Table 2).


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Table 2. Mechanical performance of IFM skinned fibers from normal and mutant flies

 

Fractional stiffness is estimated as the contribution of cross bridge-attached filaments by subtracting the relaxed stiffness from the activated stiffness (see MATERIALS AND METHODS). Table 3 shows that for wild type, Mhc6, and fln0, nearly half the total stiffness during maximal Ca2+ activation (pCa 5.0) can be attributed to working cross bridges, a contribution that is reduced by almost half in Mhc13. The cross bridge contribution to active stiffness in Mhc6 and fln0 was 70–80% of that in wild type, whereas that in Mhc13 was ~50%. For fln0 and Mhc13, the reduction in active stiffness was of the same magnitude as the reduction in passive stiffness, suggesting that the mutations are affecting the same passive elements in series with the cross bridges. In contrast, the magnitude of the stiffness decline for Mhc6 was twice as large in active conditions than in relaxing conditions, suggesting that both cross bridges and passive elements are being affected by the mutation. The similar behavior of the mutant fibers suggests that all three mutations affect thick filament stiffness.


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Table 3. Cross bridge contribution to myofilament stiffness in skinned IFM fibers

 

A fiber's dynamic stiffness modulus can be separated into its two components, the elastic modulus (Ee) and the viscous modulus (Ev). The Ee is the component that is in phase with the applied length change. Figure 2 is a plot of Ee vs. frequency for all three mutant fibers and wild-type fibers at maximal Ca2+ activation (pCa 5.0). The plot for wild-type fibers show a characteristic triphasic response indicative of a properly functioning mechanical system, with a definitive phase shift in the intermediate frequency range and minimum amplitude near the physiologically relevant range at which the frequency matches the wing beat (corrected for the experimental temperature of 15°C).



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Fig. 2. Elastic moduli of maximally Ca2+ (pCa 5.0)-activated mutant and wild-type fibers as a function of frequency. Mhc13 and fln0 show significantly reduced elastic modulus. Values are means ± SE for wild-type (n = 11), fln0 (n = 14), Mhc6 (n = 8), and Mhc13 fibers (n = 8).

 

The amplitude of the Ee was decreased for all three mutants at the frequency at which work is maximum (fmax) (Table 2). The reduction was greatest for Mhc13 (56 ± 14 vs. 306 ± 46 kN/m2 for wild type measured at fmax; P < 0.001) and least for Mhc6 (245 ± 55 kN/m2; P < 0.5). Nevertheless, the mutant plots follow a trajectory similar to that of wild type, showing phase shifts at similar frequencies, though substantially flattened for Mhc13. The absence of a prominent phase shift (seen as a dip in the wild type plot) in Mhc13 and fln0 suggests that these two mutations render the myofilament so compliant that most of the cross bridge-generated force is internally absorbed or, alternatively, the mutations interfere with cross bridge interaction with the thin filament.

Plots of the Ev, the out-of-phase component, are presented in Fig. 3. These plots essentially depict work absorbed by the fibers (positive values) and work produced by the fibers (negative values). For the wild type, there is a pronounced phase shift (i.e., change from positive to negative values) in the intermediate frequency range, reaching a minimum at 109 Hz (Fig. 3). The plots of each mutant are of a form similar to that of wild type but exhibit a substantially flattened Ev with no pronounced phase shift and little (Mhc6 and Mhc13) or no (fln0) negative viscosity. The flattened frequency dependence of Mhc13 and fln0 is reminiscent of that seen in wild-type fibers at pCa 8.0. The negative Ev at fmax is significantly decreased in all three mutants, resulting in a nearly complete loss in power output (Table 2).



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Fig. 3. Viscous moduli of maximally Ca2+ (pCa 5.0)-activated mutant and wild-type fibers as a function of frequency. All 3 mutants exhibit a significant reduction in negative viscosity. Values are means ± SE for wild-type (n = 11), fln0 (n = 14), Mhc6 (n = 8), and Mhc13 fibers (n = 8).

 

Unlike the wild type, we observed considerable variability in the work-producing and work-absorbing properties of fibers in each mutant strain. We therefore classified fibers into three groups on the basis of their net work output (Fig. 4). The first group, work-producing fibers, included those with a demonstrable negative viscosity in the plot flanking the characteristic frequency of the work-producing process. Wild-type fibers were within this group. The second group, work-absorbing fibers, included those with no demonstrable negative viscosity, i.e., the work-producing processes were not large enough to offset the work-absorbing processes, thereby producing no net work. The third group included fibers that showed no demonstrable response to the sinusoidal oscillations. All of the wild-type fibers tested produced work, in contrast to Mhc6 (63%), Mhc13 (25%), and fln0 (7%) (Fig. 4). The majority of fln0 fibers showed net work absorption, whereas most of the Mhc13 fibers did not generate work.



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Fig. 4. Fiber net work output classification based on Nyquist plots and viscous modulus. Histogram shows the percentage of fibers from each strain that produced work, absorbed work, or did not respond to sinusoidal perturbations (not working). Values are means ± SE for wild-type (n = 11), fln0 (n = 14), Mhc6 (n = 8), and Mhc13 fibers (n = 8).

 

Effect of the mutations on fiber integrity. Fibers from the three mutants behaved abnormally when activating solution was replaced with rigor solution at the conclusion of the mechanics protocol. In the majority of cases (>75%), fibers broke immediately upon exposure to rigor solution. Occasionally, fibers would swell up at one end of the T clip attachments and show signs of fraying along the longitudinal axis, eventually breaking as the fiber was being removed from the testing apparatus after the experiment. This was in sharp contrast to wild-type fibers, which never showed signs of swelling, fraying, or breaking. The abnormal responses and appearance of mutant fibers in rigor is very reminiscent of the IFM hypercontraction phenotype in vivo previously described for Mhc6, Mhc13, and fln0 (11, 26).

To determine whether damage to the mutant fibers was precipitated by the imposed mechanical perturbations, we performed an abbreviated protocol in which Ca2+-activated fibers were transferred to rigor without intermittent sinusoidal oscillations. Fibers from fln0 and Mhc13 responded to rigor in the same way, whether or not they were mechanically activated. Fibers from Mhc6 did not break in rigor after the abbreviated procedure. In summary, the structural integrity of the fiber is compromised by the mutations in flightin and MHC; in particular, fibers from fln0 and Mhc13 are more fragile than fibers from Mhc6.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
This study has shown that the passive and dynamic mechanical properties of Drosophila IFM are affected by mutations that interfere with flightin expression. Because the mutations examined in this study cause time-dependent fiber degeneration, fibers were analyzed from adults <2 h old, when mutant and wild-type fibers are similar in appearance. The magnitude of the dynamic stiffness modulus coefficient for <2-h-old adult wild-type flies is ~40–50% of that reported for 2- to 4-day-old adults (20, 21). The most likely explanation for the lower dynamic stiffness is that 2-h-old sarcomeres do not achieve the dimensions of fully matured sarcomeres and therefore have fewer thick filaments per cross-sectional area in each myofibril (25). A contributing factor may be age-related differences in the phosphorylation level of flightin and other phosphoproteins that modify the mechanical performance of the IFM (16, 28, 32). For example, 2-h-old adults generally do not contain the full complement of nine flightin phosphovariants that are normally found in more mature adults (32).

Flightin and passive stiffness of the IFM. One of the most defining characteristics of insect IFM is their high relaxed stiffness, which previous work suggest arises largely from connecting filaments (36). Experiments using gelsolin and µ-calpain (15) indicate that projectin and kettin are both constituents of connecting filaments and that both sets of proteins are major determinants of passive stiffness (12).

Kulke et al. (12) also reported that treatment of myofibrils with the proline-specific endoproteinase Igase did not affect passive stiffness. Because Igase preferentially cleaves troponin H and flightin, they concluded that neither of these two proteins contribute to passive stiffness (12). In contrast, the present study shows that passive stiffness in Mhc13 and fln0 is reduced by >50%, a difference that we ascribe to the absence of flightin, because passive stiffness in Mhc6 is not significantly different from that in wild type. One possible explanation for this apparent discrepancy between our interpretation and the conclusion of Kulke et al. is that Igase treatment may fail to digest all of flightin (in contrast to fln0, where flightin is completely absent). Another possibility is that the part of the flightin protein exposed to Igase treatment may not contribute to stiffness. Yet another possibility is that the reduction in stiffness in the mutant fibers arises from developmental defects (e.g., failure of myofilaments to align properly), rather than from any function flightin may play in the adult fiber. We consider this last possibility unlikely because the ultrastructure of the developing IFM in Mhc13 is very close to normal (11) and yet this mutant exhibits the largest decrease in passive stiffness.

The decrease in dynamic stiffness of the mutants parallels the decrease in passive stiffness, suggesting that the latter contributes to the former. These results are consistent with the proposal that the stiffness of the relaxed muscle contributes to that of the active muscle (36). A prevailing trend in this study is that the passive and dynamic stiffnesses of Mhc13 closely mimic those of fln0, whereas the stiffnesses of Mhc6 mimic those of wild type. In line with this trend, the in vivo phenotype of Mhc13 is more similar to that of fln0 than to that of Mhc6 (11, 26). We attribute this correspondence to the effect the mutations have on flightin expression. Studies in vitro showing that the mutation in Mhc13 abolishes flightin binding to the myosin rod provide a likely explanation for why Mhc13 IFM has little or no flightin (1). At <2 h of adult age, the expression profile of flightin (11) and the passive stiffness in Mhc6 are very similar to those of wild type. One remarkable feature is that the passive tension in Mhc6 is slightly but significantly increased relative to wild type (Fig. 1). This difference could be due to the relative expression of the different flightin isoelectric variants. In particular, we note that the ratio of nonphosphorylated (N1) to phosphorylated flightin is more than twice as high in Mhc6 than in wild type [(11) and unpublished observations].

Alterations in flightin expression affect stretch activation. Stretch activation underlies the ability of striated muscle to perform oscillatory work. In sinusoidal analysis this is manifested as a prominent –Ev, the magnitude of which is proportional to the work produced by the fiber. All three mutants exhibited significant reductions in the amplitude of Ev. As shown in Fig. 3, the graded effect of the mutations on the amplitude of Ev parallels their effect on flightin expression, with fln0 showing the greatest reduction in flightin expression and Ev. The reduced ability or inability of the mutant fibers to produce oscillatory work could arise from defects in force production, force transmission, or a combination of both. However, except for Mhc13, maximal isometric tension (pCa 5) of each mutant was not significantly different from that of wild type, suggesting that the mutations primarily affect force transmission. We reach this conclusion because maximal isometric tension (relatively unaffected) does not depend on the viscous properties of the filaments transmitting force to the ends of the sarcomere, whereas oscillatory work (dramatically affected) does.

It is reasonable to assume that altering or deleting flightin affects the viscoelasticity of the thick filament, given the distribution of this protein throughout the A band and its association with the myosin rod (1, 26, 33). On the basis of changes in skinned fiber stiffness, we speculate that both the Ee and Ev of the thick filament are reduced. If so, it is possible to account for isometric force levels comparable to wild type if the effect of additional strain in the thick filament is offset by an associated reduction in I-band width at steady-state isometric tension. Likewise, it is possible to account for a reduced oscillatory work output if the mutant filaments are less viscous.

The increased Ca2+ sensitivity of isometric tension in Mhc13 could be due to closer proximity of the myosin heads to actin. There is no a priori reason to believe that a mutation of flightin on the thick filament directly affects interfilament lattice spacing. However, it is possible that modification or removal of the flightin molecule allows the thick filament to expand, thereby moving the heads closer to actin. Previous studies in skeletal and cardiac fibers have shown that movement of the myosin heads closer to actin by manipulating interfilament spacing increases Ca2+ sensitivity of tension (7, 17). For example, when fibers at rest length are osmotically compressed with 2.5% dextran, the pCa-tension relation shifts leftward, a shift that is roughly equivalent to that observed when filament spacing is reduced by approximately the same extent by stretching the sarcomere (18). It should be noted, however, that recent studies suggest that the effect of dextran (osmotic compression) on Ca2+ sensitivity is likely to be independent of changes in myofilament spacing (10a).

Both myosin mutations studied here cause a leftward shift of the force-pCa relation (i.e., increased pCa50) and a decrease in the slope of the relation (lower Hill coefficient), an effect reminiscent of that observed in skeletal fibers at increasing sarcomere lengths (17). Thus it is tempting to speculate that the rod mutations loosen the packing density of the thick filament, causing the heads to move closer to actin as the thick filament diameter increases and thereby increasing the probability of myosin head interaction with actin and S1-dependent activation of the thin filament (7). The more pronounced effect of Mhc13 could be due to a more pronounced effect on thick filament diameter due to the charge change introduced by this mutation (glutamic acid to lysine). In contrast, Mhc6 (arginine to histidine) preserves the charge state (11), so the effect on filament diameter would be correspondingly less, as observed. These results suggest that thick filament charge is an important component of the effect of lattice spacing on Ca2+ sensitivity (17).

One important distinction among the mutants is their effect on the Ee. Mhc13 and fln0 result in a significant reduction in Ee, whereas Mhc6 does not, suggesting that small alterations in flightin phosphorylation have little or no effect on fiber elasticity. The interaction of flightin with other thick filament proteins, rather than its phosphorylation status, may be more important in determining the elastic properties of the fiber. In contrast, the dynamic Ev appears to be sensitive to alterations in the phosphorylation status of flightin (i.e., the ratio of nonphosphorylated to phosphorylated flightin), raising the possibility that oscillatory power production could be modulated by changes in flightin phosphorylation.

Functional properties of flightin in Drosophila IFM. Several studies have demonstrated that a significant portion of sarcomere compliance resides in thin filaments and the thick filament backbone (34). Variations in myofilament compliance could be one way of modulating the amplitude and kinetics of force production among muscle types (15). Insect IFM myofibrils have a much higher stiffness than vertebrate skeletal and cardiac muscles (12, 36). This is believed to have a mechanical effect on the kinetics of cross bridge activation and account for the enhanced delayed force response to stretch of the IFM (30).

A recent study showed that fiber hypercontraction in Mhc13 and fln0 can be suppressed by substituting myosin with a "headless" transgene, i.e., a myosin construct that is missing the motor domain but can assemble thick filaments (22). These results indicate that contractile forces are responsible for the sarcomere breakdown and fiber bunching that characterizes the myosin rod and flightin mutants, a fact further supported by our observations in this study that mutant skinned fibers lose their structural integrity upon exposure to rigor solution. Absence of flightin does not impair rigor cross bridge formation (26) and, as shown here, does not impair force production under isometric conditions. We interpret the reduction in resting stiffness, in vivo hypercontraction, and rigor-induced fiber breakage as different manifestations of the same condition, namely, a sarcomeric lattice that is compromised by the absence of a critical structural element. On the basis of gel analysis of IFM proteins from Mhc13 and Mhc6, flightin is the only protein whose expression appears to be affected by these two mutations (11, 19). We propose that flightin stiffens the thick filament and is an essential structural element for the enhanced delayed force response to stretch and elastic energy storage.

Changes in myofilament compliance can explain the differences in passive and dynamic properties that are manifested in the mutant fibers. Alterations in the compliance of thick filaments can directly account for the changes in Ee and indirectly for the changes in Ev and fmax, given that the properties of cycling cross bridges are sensitive to the properties of series elastic elements (see e.g., Ref. 15). Because much of the work produced by fln0 fibers is internally absorbed, the contribution of flightin to filament stiffness (by reducing filament viscoelasticity) appears to be essential for efficient transmission of contractile forces.

There are several possibilities for how flightin may influence myofibril stiffness and work output. One possibility is that the absence or reduction of flightin affects the interaction of projectin and/or kettin with the thick filament. However, this aberrant interaction must result from an indirect effect, because kettin and projectin emanate from the Z band and extend only to the I/A junction, a region of the sarcomere where flightin is not normally found (12, 26).

Another, more likely, possibility is that flightin contributes to thick filament stiffness by cross-linking myosin pairs into subfilaments and/or by direct interaction with the S2 hinge (26). The binding of flightin to the LMM (1) and the susceptibility of the S2 hinge to proteolysis in Mhc13 (11) and fln0 (26) lend support to this proposal.

It is also possible that flightin forms part of a structure that connects the thick filament to the thin filament. The reduction in elastic stiffness modulus seen in Mhc13 and fln0 is consistent with this interpretation. Removal of the NH2-terminal extension of the Drosophila myosin regulatory light chain, which has been proposed to form a parallel link between thick and thin filaments, also resulted in decreased Ee (20). Furthermore, Tawada and Kawai (29) have shown that cross-linking rabbit psoas fibers with 1-ethyl-3-[3-(dimethylamino)proyl]carbodiimide resulted in an enhanced stretch activation response that mimicked the kinetics of insect flight muscle. Thus the high passive stiffness and the enhanced delayed force response to stretch of IFM could be explained, at least in part, by the presence of thin filament to thick filament cross-linking structures.

In summary, we have shown that IFM fibers from the mutant strains Mhc13 and fln0 are substantially more compliant than wild type and that, as a result, the oscillatory work production is completely abolished in these mutants. Fibers from the Mhc6 mutant show a severe reduction in work production, but with little or no accompanying change in compliance. These results suggest that flightin contributes significantly to the passive mechanical properties of insect IFM and is essential for normal work and power output. Furthermore, because flight efficiency is remarkably low in Drosophila and in other insects with asynchronous flight muscles [in part a reflection of the low efficiency by which these muscles convert chemical energy into mechanical power (5, 13)], energy recuperation through flightin-enhanced elastic storage may be an important aid for flying flies (5). The expression of flightin in the flight muscles can thus be seen as a partial solution to the problem of minimizing the energetic cost of flight and as a novel step in the evolution of flight systems.


    ACKNOWLEDGMENTS
 
We thank William Barnes and Allison Cox for technical support for this project, Alan Howard for contributing statistical expertise, and Mark Miller, Jeff Moore, Brad Palmer, and Doug Swank for assistance in data analysis, interpretation, and comments for this manuscript.

GRANTS

This work was supported by National Science Foundation Grants MCB0090768 and MCB0315865 (to J. O. Vigoreaux).


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. Henkin, Dept. of Biology, Univ. of Vermont, Burlington, VT 05405 (E-mail: jhenkin{at}zoo.uvm.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
1. Ayer G and Vigoreaux JO. Flightin is a myosin rod binding protein. Cell Biochem Biophys 38: 41–54, 2003.[ISI][Medline]

2. Benian GM, Ayme-Southgate A, and Tinley TL. The genetics and molecular biology of the titin/connectin-like proteins of invertebrates. Rev Physiol Biochem Pharmacol 138: 235–268, 1999.[ISI][Medline]

3. Chan WP and Dickinson MH. In vivo length oscillations of indirect flight muscles in the fruit fly Drosophila virilis. J Exp Biol 199: 2767–2774, 1996.[Abstract/Free Full Text]

4. Dickinson MH, Hyatt CJ, Lehmann FO, Moore JR, Reedy MC, Simcox A, Tohtong R, Vigoreaux JO, Yamashita H, and Maughan DW. Phosphorylation-dependent power output of transgenic flies: an integrated study. Biophys J 73: 3122–3134, 1997.[Abstract]

5. Dickinson MH and Lighton JRB. Muscle efficiency and elastic storage in the flight motor of Drosophila. Science 268: 87–90, 1995.[ISI][Medline]

6. Godt RE and Lindley BD. Influence of temperature upon contractile activation and isometric force production in mechanically skinned muscle fibers of the frog. J Gen Physiol 80: 279–297, 1982.[Abstract]

7. Gordon AM, Homsher E, and Regnier M. Regulation of contraction in striated muscle. Physiol Rev 80: 853–924, 2000.[Abstract/Free Full Text]

8. Granzier HLM and Wang K. Interplay between passive tension and strong and weak binding cross-bridges in insect indirect flight muscle. J Gen Physiol 101: 235–270, 1993.[Abstract]

9. Josephson RK. Contraction dynamics and power output of skeletal muscle. Annu Rev Physiol 55: 527–546, 1993.[CrossRef][ISI][Medline]

10. Josephson RK, Malamud JG, and Stokes DR. Asynchronous muscle: a primer. J Exp Biol 203: 2713–2722, 2000.[Abstract/Free Full Text]

10. Konhilas JP, Irving TC, and Tombe PP. Length-dependent activation in three striated muscle types of the rat. J Physiol 544: 225–236, 2002.[Abstract/Free Full Text]

11. Kronert WA, O'Donnell PT, Fieck A, Lawn A, Vigoreaux JO, Sparrow JC, and Bernstein SI. Defects in the Drosophila myosin rod permit sarcomere assembly but cause flight muscle degeneration. J Mol Biol 249: 111–125, 1995.[CrossRef][ISI][Medline]

12. Kulke M, Neagoe C, Kolmerer B, Minajeva A, Hinssen H, Bullard B, and Linke WA. Kettin, a major source of myofibrillar stiffness in Drosophila indirect flight muscle. J Cell Biol 154: 1045–1057, 2001.[Abstract/Free Full Text]

13. Lehmann FO and Dickinson MH. The production of elevated flight force compromises manoeuvrability in the fruit fly Drosophila melanogaster. J Exp Biol 204: 627–635, 2001.[Abstract/Free Full Text]

14. Linke WA and Granzier H. A spring tale: new facts on titin elasticity. Biophys J 75: 2613–2614, 1998.[Free Full Text]

15. Luo Y, Cooke R, and Pate E. A model of stress relaxation in cross-bridge systems: effect of a series elastic element. Am J Physiol Cell Physiol 265: C279–C288, 1993.[Abstract/Free Full Text]

16. Maroto M, Arredondo J, Goulding D, Marco R, Bullard B, and Cervera M. Drosophila paramyosin/miniparamyosin gene products show a large diversity in quantity, localization, and isoform pattern: a possible role in muscle maturation and function. J Cell Biol 134: 81–92, 1996.[Abstract]

17. Martyn DA and Gordon AM. Length and myofilament spacing-dependent changes in calcium sensitivity of skeletal fibres: effects of pH and ionic strength. J Muscle Res Cell Motil 9: 428–445, 1988.[ISI][Medline]

18. McDonald KS and Moss RL. Osmotic compression of single cardiac myocytes eliminates the reduction in Ca2+ sensitivity of tension at short sarcomere length. Circ Res 77: 199–205, 1995.[Abstract/Free Full Text]

19. Mogami K, Fujita SC, and Hotta Y. Identification of Drosophila indirect flight muscle myofibrillar proteins by means of two-dimensional electrophoresis. J Biochem (Tokyo) 91: 643–650, 1982.[Abstract]

20. Moore JR, Dickinson MH, Vigoreaux JO, and Maughan DM. The effect of removing the N-terminal extension of the Drosophila myosin regulatory light chain upon flight ability and the contractile dynamics of indirect flight muscles. Biophys J 78: 1431–1440, 2000.[Abstract/Free Full Text]

21. Moore JR, Vigoreaux JO, and Maughan DW. The Drosophila projectin mutant, bentD, has reduced stretch activation and altered indirect flight muscle kinetics. J Muscle Res Cell Motil 20: 797–806, 1999.[CrossRef][ISI][Medline]

22. Nongthomba U, Cummins M, Clark S, Vigoreaux JO, and Sparrow JC. Suppression of the muscle hypercontraction phenotype by mutations in the myosin heavy chain gene of Drosophila melanogaster. Genetics 164: 209–222, 2003.[Abstract/Free Full Text]

23. Peckham M, Molloy JE, Sparrow JC, and White DCS. Physiological properties of the dorsal longitudinal flight muscle and the tergal depressor of the trochanter muscle of Drosophila melanogaster. J Muscle Res Cell Motil 11: 203–215, 1990.[ISI][Medline]

24. Pringle JWS. The Croonian Lecture, 1977. Stretch activation of muscle: function and mechanism. Proc R Soc Lond B Biol Sci 201: 107–130, 1978.[ISI][Medline]

25. Reedy MC and Beall C. Ultrastructure of developing flight muscle in Drosophila. I. Assembly of myofibrils. Dev Biol 160: 443–465, 1993.[CrossRef][ISI][Medline]

26. Reedy MC, Bullard B, and Vigoreaux JO. Flightin is essential for thick filament assembly and sarcomere stability in Drosophila flight muscles. J Cell Biol 151: 1483–1499, 2000.[Abstract/Free Full Text]

27. Saide JD. Identification of a connecting filament protein in insect fibrillar flight muscle. J Mol Biol 153: 661–679, 1981.[ISI][Medline]

28. Takano-Ohmuro H, Takahashi S, Hirose G, and Maruyama K. Phosphorylated and dephosphorylated myosin light chains of Drosophila fly and larva. Comp Biochem Physiol B 95: 171–177, 1990.[ISI][Medline]

29. Tawada K and Kawai M. Covalent cross-linking of single fibers from rabbit psoas increases oscillatory power. Biophys J 57: 643–647, 1990.[Abstract]

30. Thorson J and White DCS. Role of cross-bridge distortion in the small-signal mechanical dynamics of insect and rabbit skeletal muscle. J Physiol 343: 59–84, 1983.[Abstract]

31. Vigoreaux JO. Genetics of the Drosophila flight muscle myofibril: a window into the biology of complex systems. Bioessays 23: 1047–1063, 2001.[CrossRef][ISI][Medline]

32. Vigoreaux JO and Perry LM. Multiple isoelectric variants of flightin in Drosophila stretch-activated muscles are generated by temporally regulated phosphorylations. J Muscle Res Cell Motil 15: 607–616, 1994.[ISI][Medline]

33. Vigoreaux JO, Saide JD, Valgeirsdottir K, and Pardue ML. Flightin, a novel myofibrillar protein of Drosophila stretch-activated muscles. J Cell Biol 121: 587–598, 1993.[Abstract]

34. Wakabayashi K, Sugimoto Y, Tanaka H, Ueno Y, Takezawa Y, and Amemiya Y. X-ray diffraction evidence for the extensibility of actin and myosin filaments during muscle contraction. Biophys J 67: 2422–2435, 1994.[Abstract]

35. Wang K, McCarter R, Wright J, Beverly J, and Ramirez-Mitchell R. Regulation of skeletal muscle stiffness and elasticity by titin isoforms: a test of the segmental extension model of resting tension. Proc Natl Acad Sci USA 88: 7101–7105, 1991.[Abstract]

36. White DCS. The elasticity of relaxed insect fibrillar flight muscle. J Physiol 343: 31–57, 1983.[Abstract]