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Mol Biol Cell. 2008 February; 19(2): 553–562.
PMCID: PMC2230588

Myosin Transducer Mutations Differentially Affect Motor Function, Myofibril Structure, and the Performance of Skeletal and Cardiac Muscles

Thomas Pollard, Monitoring Editor

Abstract

Striated muscle myosin is a multidomain ATP-dependent molecular motor. Alterations to various domains affect the chemomechanical properties of the motor, and they are associated with skeletal and cardiac myopathies. The myosin transducer domain is located near the nucleotide-binding site. Here, we helped define the role of the transducer by using an integrative approach to study how Drosophila melanogaster transducer mutations D45 and Mhc5 affect myosin function and skeletal and cardiac muscle structure and performance. We found D45 (A261T) myosin has depressed ATPase activity and in vitro actin motility, whereas Mhc5 (G200D) myosin has these properties enhanced. Depressed D45 myosin activity protects against age-associated dysfunction in metabolically demanding skeletal muscles. In contrast, enhanced Mhc5 myosin function allows normal skeletal myofibril assembly, but it induces degradation of the myofibrillar apparatus, probably as a result of contractile disinhibition. Analysis of beating hearts demonstrates depressed motor function evokes a dilatory response, similar to that seen with vertebrate dilated cardiomyopathy myosin mutations, and it disrupts contractile rhythmicity. Enhanced myosin performance generates a phenotype apparently analogous to that of human restrictive cardiomyopathy, possibly indicating myosin-based origins for the disease. The D45 and Mhc5 mutations illustrate the transducer's role in influencing the chemomechanical properties of myosin and produce unique pathologies in distinct muscles. Our data suggest Drosophila is a valuable system for identifying and modeling mutations analogous to those associated with specific human muscle disorders.

INTRODUCTION

The myosin molecular motor of striated muscle is a hexameric molecule composed of two myosin heavy chains (MHCs) and four light chains. The N-terminal globular motor domain is a product inhibited ATPase comprised of several communicating domains and functional units (reviewed by Geeves and Holmes, 1999 blue right-pointing triangle, 2005 blue right-pointing triangle; Geeves et al., 2005 blue right-pointing triangle). Alterations to the various domains dramatically affect the biochemical and mechanical properties of the motor in vitro, and mutations that diminish or enhance the molecular performance of myosin in vivo are associated with the pathogenesis of both skeletal and cardiac myopathies (reviewed by Sellers, 1999 blue right-pointing triangle; Redowicz, 2002 blue right-pointing triangle; Oldfors et al., 2004 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle; Laing and Nowak, 2005 blue right-pointing triangle; Tardiff, 2005 blue right-pointing triangle; Oldfors, 2007 blue right-pointing triangle). Central to an understanding of myosin-based myopathies is a fundamental appreciation for how depressed or enhanced molecular motor function differentially affects diverse striated muscles.

Among the communicating functional units of myosin is the recently described transducer region (Coureux et al., 2004 blue right-pointing triangle). Myosin V crystal structures reveal the transducer's central location within the motor domain near the nucleotide binding site. The transducer includes the last three strands of a seven-stranded β-sheet, which undergoes distortion essential for rearrangements within the nucleotide pocket during the ATPase cycle, and the loops and linkers that accommodate the distortion (Coureux et al., 2004 blue right-pointing triangle). Among the elements of the transducer is hypervariable loop 1, a flexible surface loop shown to influence a wide range of kinetic and mechanical properties of myosin (Kurzawa-Goertz et al., 1998 blue right-pointing triangle; Sweeney et al., 1998 blue right-pointing triangle; Clark et al., 2005 blue right-pointing triangle). The transducer ultimately integrates information from all parts of the motor domain to facilitate efficient conversion of the energy liberated during ATP hydrolysis into force production (Coureux et al., 2004 blue right-pointing triangle).

In striated muscle, myosin-containing thick filaments drive contraction in an ATP-dependent manner through cyclical interactions with actin-containing thin filaments. The thin filament troponin–tropomyosin regulatory complex inhibits contraction in resting muscle by occluding myosin binding sites on actin (reviewed by Gordon et al., 2000 blue right-pointing triangle; Brown and Cohen, 2005 blue right-pointing triangle). On activation, tropomyosin shifts positions in a stepwise manner away from these binding sites, first as a result of Ca2+ binding to troponin and then by myosin cross-bridge binding to actin (McKillop and Geeves, 1993 blue right-pointing triangle; Vibert et al., 1997 blue right-pointing triangle; Poole et al., 2006 blue right-pointing triangle). Initial cross-bridge binding to actin seems to have allosteric effects on thin filaments such that there is a spread of accessible myosin binding sites along the filaments leading to cooperative activation of contraction (reviewed by Tobacman, 1996 blue right-pointing triangle; Gordon et al., 2000 blue right-pointing triangle; Moss et al., 2004 blue right-pointing triangle). Thus, both elevated Ca2+ levels and cross-bridge binding are required for full activation.

Investigations of the functional domains of myosin and the effects of mutations on muscle contractile properties are greatly facilitated by studying the genetically tractable Drosophila melanogaster system. The Drosophila Mhc gene exists as a single copy per haploid genome that encodes all muscle MHCs through alternative splicing of the primary transcript (Bernstein et al., 1983 blue right-pointing triangle; Rozek and Davidson, 1983 blue right-pointing triangle; George et al., 1989 blue right-pointing triangle). Nonlethal mutations located in constitutive exons are expressed in all myosin isoforms of every striated muscle. Consequently, in such mutants, unlike in vertebrate systems possessing complex Mhc multigene families, compensatory up-regulation of nonmutated myosin isoforms cannot occur. Furthermore, changes in striated muscle performance due to developmental or senescent-dependent switches in myosin isoform complements lacking the mutations are impossible.

The effects of particular myosin isoforms on muscle function during aging can be readily studied in Drosophila. Muscles amenable to such analyses include indirect fight (IFM) (Baker, 1976 blue right-pointing triangle; Magwere et al., 2006 blue right-pointing triangle) and cardiac muscles (Paternostro et al., 2001 blue right-pointing triangle; Wolf et al., 2006 blue right-pointing triangle; Ocorr et al., 2007 blue right-pointing triangle). IFM function is not required for viability, and Drosophila cardiac function can be dramatically compromised without causing immediate death. Drosophila age in weeks, and they share common mechanisms that determine aging rates and longevity with higher organisms (Parkes et al., 1999 blue right-pointing triangle; Finch and Ruvkun, 2001 blue right-pointing triangle; Tatar et al., 2003 blue right-pointing triangle; Wessells and Bodmer, 2007 blue right-pointing triangle). Thus, the fly is an extremely powerful model for studying the progression of myosin-related skeletal and cardiac muscle dysfunction.

Two myosin point mutations in the Drosophila Mhc gene, D45 and Mhc5, were localized in proximity to coding regions for loops and linkers of the transducer (Kronert et al., 1999 blue right-pointing triangle; Montana and Littleton, 2004 blue right-pointing triangle). These mutations are in Mhc constitutive exons 5 and 4, respectively. Both mutations impair Drosophila flight ability, and they either suppress (D45) or enhance (Mhc5) IFM myofibrillar destruction when combined with certain troponin mutations (Kronert et al., 1999 blue right-pointing triangle). These findings suggest the amino acid changes differentially alter the fundamental chemomechanical properties of myosin and possibly mimic perturbations in motor function caused by certain myosin-based myopathy mutations. Because every isoform of striated muscle myosin from the two lines possesses the alterations, the mutants are unique tools for examining the pathophysiological responses to perturbed motor function in distinct striated muscles of a single model organism.

Here, we provide the first detailed analysis of the effects of mutations in specific transducer elements on myosin molecular function and on age-related changes in skeletal and cardiac muscles. To identify changes in molecular motor performance, myosin was purified from IFM of wild-type and mutant flies for biochemical and biophysical analyses. Skeletal muscle locomotory function was assessed by evaluating flight abilities of wild-type and transducer mutant flies throughout life. We used electron microscopy to evaluate the consequences of mutant myosin expression on IFM myofibrillar ultrastructure. We additionally investigated the progressive changes in cardiac structure and function as a result of altered motor properties by using high-speed video microscopy and advanced motion detection analysis. Our efforts to define the role of the transducer in determining chemomechanical properties of myosin and in diverse striated muscles demonstrate that Drosophila is useful for investigating the pathogenesis of skeletal and cardiac disorders. Furthermore, our model may serve to identify novel mutations that lead to specific myopathies found in the human population.

MATERIALS AND METHODS

Fly Lines and Aging

yw (wild-type), D45, and Mhc5 Drosophila were maintained on a standard yeast-sucrose-agar medium at 25°C. For flight and cardiac muscle studies, newly eclosed flies were collected over an 8-h span. All flies were transferred to fresh vials every 2–3 d over a 5-wk period.

Protein Isolation and Purification

IFM myosin was purified from 1- to 2-d-old yw or mutant flies as described previously (Swank et al., 2001 blue right-pointing triangle) with the following modifications: 10 mM dithiothreitol (DTT) in distilled H2O was used for all dilutions, and all solution volumes were decreased to 75% of previously published volumes. Purified myosin pellets were resuspended in myosin storage buffer consisting of 0.5 M KCl, 20 mM 3-(N-morpholino)propanesulfonic acid, pH 7.0, 2 mM MgCl2, and 20 mM DTT. Subsequent biochemical and mechanical experiments were performed immediately after spectroscopic methods (Margossian and Lowey, 1982 blue right-pointing triangle) for determining myosin concentrations.

ATPase Assays

Myosin ATPase activities were determined using [γ-32P]ATP. Ca2+ ATPase was measured as described previously (Swank et al., 2001 blue right-pointing triangle). Actin-activated ATPase was determined using chicken skeletal muscle actin (Swank et al., 2003 blue right-pointing triangle). G-actin was isolated from acetone powder of chicken skeletal muscle according to Pardee and Spudich (1982) blue right-pointing triangle. F-actin was prepared by adding 1 volume of 10X polymerization buffer (50 mM Tris-Cl, pH 8, 0.5 M KCl, 20 mM MgCl2, and 10 mM ATP) to 9 volumes of G-actin. The working F-actin solution had a concentration of ~30 μM, so that the amount of nonradioactive ATP added to the reaction mixture was minimized. Actin-activated Vmax and Km values for actin were computed by fitting all data points from multiple preparations of each myosin isoform (wild type [n = 9], D45 [n = 4], or Mhc5 [n = 3]) with the Michaelis-Menten equation. Values were averaged to give mean ± SD. Statistical differences in Vmax and Km between wild-type and mutant myosin isoforms were evaluated using Student's t tests.

In Vitro Motility Assays

In vitro actin sliding velocity was determined according to Swank et al. (2001) blue right-pointing triangle, with some alterations: 20 mM DTT was used in all solutions of the assay. The 0 salt motility assay buffer/0.4% methyl cellulose/glucose oxidase and catylase (0B/MC/GOC) and 0B/MC/GOC + ATP (0B/MC/GOC with 2 mM ATP added) solutions were diluted to 70% of that used previously. Lower ionic strength elevated levels of continuous movement for the majority of actin filaments. Analysis of captured video sequences was performed as described by Root and Wang (1994) blue right-pointing triangle, by using the modifications described in Swank et al. (2001) blue right-pointing triangle. Velocities of 15–20 individual filaments were calculated and recorded from each assay; values from multiple preparations (wild-type [n = 4], D45 [n = 4], or Mhc5 [n = 3]) were averaged to give mean ± SD. Statistical differences in the average velocity of actin filaments driven by wild-type and mutant myosin isoforms were determined by Student's t tests.

Flight Testing

Flight testing of 2-, 7-, 21-, or 35-d-old flies was performed as described in Drummond et al. (1991) blue right-pointing triangle and Suggs et al. (2007) blue right-pointing triangle. Each fly was assigned a flight index (FI) value based on its ability to fly up (6), horizontal (4), downward (2), or not at all (0). The average FI (±SD) for each line was calculated by dividing the sum of the individual FI values by the number of individuals tested (n > 200) at each age point. Statistical testing of age-associated changes in mean FI among groups and within each line was performed as described below for calculating differences in age-dependent changes in cardiac parameters between and within genotypes.

Electron Microscopy of Drosophila IFM

Thoraces from late pupa and 2-d-old female flies were isolated and prepared for transmission electron microscopy according to Cripps et al. (1994) blue right-pointing triangle. Fixatives and Embed812 resin were purchased from Electron Microscopy Sciences (Fort Washington, PA); other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Late pupal and 2-d-old adult samples were examined on a Philips 410 transmission electron microscope operating at 80 kV. Images were recorded on film and later digitized using an Epson 1640SU PHOTO flatbed scanner. Young adult and some pupal samples were examined with a FEI Tecnai 12 transmission electron microscope operating at 120 kV. Digital images were taken with a TemCam-F214 high-resolution digital camera (TVIPS-Tietz, Gauting, Germany). Microscope magnifications were calibrated using a diffraction grating replica and latex calibration standard (Ted Pella, Redding, CA).

Image Analysis of Semi-intact Heart Preparations

Image analysis of beating, semi-intact heart preparations from 1-, 2-, 3-, 4-, and 5-wk-old adults was performed according to Ocorr et al. (2007) blue right-pointing triangle. M-modes were generated using a MatLab (MathWorks, Natick, MA)-based image analysis program (Ocorr et al., 2007 blue right-pointing triangle). Briefly, a 1 pixel-wide region is defined in a single frame of a high-speed digital movie that encompasses both edges of the heart tube; identical regions are then cut from all consecutive movie frames and aligned horizontally. This provides an edge trace that documents the movement of the heart tube walls in the y-axis over time in the x-axis.

Measurements of diastolic and systolic diameters were made within the third abdominal segment of heart tubes directly from individual video frames. These and other cardiac contractile parameters were obtained as output from the MatLab-based program. Heart periods (HP) are defined as the time between the ends of two consecutive diastolic intervals. The arrythmicity index (AI) was calculated as the standard deviations of all recorded HP for an individual fly normalized to the median HP for that fly. Large standard deviations in HP for a single fly are a reflection of nonuniformly rhythmic contraction/relaxation cycles.

Age-dependent changes in cardiac parameters were modeled hierarchically. For all analyses, values of p < 0.05 were considered significant. Measured parameters included cardiac diameters (diastolic and systolic wall distances), percentage of fractional shortening, HP, diastolic intervals (DIs) and systolic intervals (SIs), and AI. For each fly line, we initially fit a linear model to the mean parameter values from 1 through 5 wk of age. We estimated potential nonlinear change through time using an added sums of squares F-test. For all cardiac parameters, except diastolic distances, there was no strong evidence (p > 0.05) of significant nonlinear change through time. Although diastolic heart wall distance changes with age did reveal statistical evidence of nonlinearity (p = 0.03), the deviation was considered minor; therefore, these and all other response variables were fit with linear models. Analysis of covariance (ANCOVA) was used to test for heterogeneities in the slopes of the fitted lines from each genotype, for each parameter. When significant heterogeneity in slopes was found, we estimated the different functional relationships (slopes) by regression analysis, and we tested for significant change with respect to age. If no significant heterogeneity was found among the slopes, a common slope was estimated via linear regression. We determined whether the common slope, shared among groups, was significant and we performed multiple comparisons among the elevations of the lines to investigate statistical differences between the genotypes. Analysis of variance (ANOVA) for genotype as a function of SI was used to test whether significant differences between yw and Mhc5 existed at each of the five age points.

RESULTS

D45 and Mhc5 Mutations Reside within Distinct Transducer Elements

The D. melanogaster Mhc mutants D45 and Mhc5 contain single amino acid conversions previously localized to the vicinity of ATP entry and the ATP binding site on chicken skeletal myosin S1 fragment (Kronert et al., 1999 blue right-pointing triangle). It was postulated that both mutations affect the ATPase cycle by regulating nucleotide entry or product release from the binding pocket (Kronert et al., 1999 blue right-pointing triangle). Mapping the point mutations onto chicken myosin V motor domain structures reveals that the specific amino acid alterations reside within distinct structural elements of the transducer region (Figure 1A) (Coureux et al., 2003 blue right-pointing triangle, 2004 blue right-pointing triangle). The D45 (A261T) mutation occurs at the junction between the β-bulge and the seventh strand of the central β-sheet, whereas the Mhc5 mutation (G200D) maps close to the junction between the HF helix and loop 1. Sequence comparisons corroborate the locations of the Drosophila mutations relative to specific transducer elements from different organisms (Figure 1B). The alignments also demonstrate the variability found within loop 1 primary structures among various myosin isoforms. The structural elements of the transducer were proposed to interact in a coordinated manner to bring about kinetic tuning of product release after ATP hydrolysis (Coureux et al., 2004 blue right-pointing triangle). Point mutations within the transducer may alter rates of transitions between states of the actomyosin ATPase cycle and thereby influence chemomechanical transduction in the muscles in which they are expressed.

Figure 1.
Locations of myosin transducer mutations and the alignment and sequence comparison of transducer elements from various myosin isoforms. (A) Ribbon diagram (prepared using the KiNG 1.39 [Kinemage, Next Generation, Durham, NC] interactive system for three-dimensional ...

Biochemical and Mechanical Properties of the Mutant Myosin Isoforms

To determine the effects of distinct transducer mutations on the biochemical and molecular mechanical properties of myosin in vitro, we measured Ca2+, Mg2+, and actin-stimulated ATPase activities, determined Km values for actin, and performed in vitro motility assays on the wild-type indirect flight isoform (IFI) and mutant IFM myosin (see Table 1). Basal Ca2+ ATPase activities of purified D45 and Mhc5 myosin were similar to the IFI. However, basal Mg2+ ATPase activity for D45 myosin was about half that of the IFI, whereas basal Mg2+ ATPase activity for Mhc5 myosin was more than twofold higher than for the IFI. Maximum actin-activated ATPase activity (Vmax) (Figure 2A) for D45 myosin was almost threefold lower than that of the IFI, and it was approximately twofold lower than that of Mhc5 myosin. Km, a measure of actin-binding affinity, for either transducer mutant did not differ significantly from that for wild-type myosin. In vitro motility assays (Table 1 and Figure 2B), performed using D45 myosin displayed a reduction in actin filament translocation to about half the velocity of the IFI. Myosin containing the Mhc5 mutation increased actin velocity roughly 15%. Thus, distinct mutations in the transducer region elicit opposing effects on myosin. The D45 mutation depresses the biochemical and mechanical functions of the motor, whereas the Mhc5 mutation enhances them.

Table 1.
Wild-type (IFI) and mutant myosin biochemical and mechanical data
Figure 2.
Effects of myosin transducer mutations on molecular and skeletal muscle function. (A) Steady-state rates of actin-activated myosin ATPase for IFI (●), D45 (▲) and Mhc5 (■) myosin molecules. ATPase results are represented with data ...

Effects of Mutant Myosin on Skeletal Muscle Function

Because the D. melanogaster transducer is conserved in every striated muscle myosin isoform, we investigated the effects of depressed or enhanced molecular motor function on distinct muscles with differing physiological roles and properties. We assessed skeletal muscle function by evaluating age-dependent changes in flight ability. Wild-type (yw) flies expressing IFI myosin showed a characteristically rapid and steady decline in flight ability from 2 d through 5 wk of age (Figure 2C). Over 35 d, there was an ~60% overall decrease in FI. In addition to exhibiting an ~20% reduction in initial flight ability, relative to yw, D45 flies also showed a significant decrease in FI over the 35-d period. However, the rate of decline was less and the overall decrease was only ~30%, which was markedly lower than wild type. Thus, hypoactive flight muscle myosin expressed in D45 flies provided protection against normal age-dependent muscle dysfunction. Conversely, flies expressing hyperactive Mhc5 myosin showed no flight ability at any age.

Effects of Mutant Myosin on Skeletal Muscle Ultrastructure

To ascertain whether expression of myosin with depressed or enhanced ATP hydrolytic and mechanical properties produced ultrastructural abnormalities within skeletal muscles, we examined the IFM by transmission electron microscopy (Figure 3, A–D). Longitudinal and transverse sections revealed that D45 myosin expression (Figure 3B) did not disrupt the assembly or integrity of myofibrils within the IFM, because their general appearance closely resembled that of yw myofibrils (Figure 3A). Sarcomeric structure in both lines seemed highly organized with easily discernible Z-lines, I-bands, A-bands, H-zones, and M-lines. In cross section, yw and D45 myofibrils displayed the normal, crystalline-like double hexagonal array of myofilament packing. Myofibrils containing the Mhc5 isoform seem to have assembled normally, as shown by longitudinal and cross sections through late pupae (Figure 3C). By 2 d after eclosion, however, adult Mhc5 IFM displayed a dramatic disarray of contractile filaments with a loss of sarcomeric structure and integrity of Z-lines, I-bands, A-bands, H-zones, and M-lines (Figure 3D). This is reminiscent of the IFM phenotype arising from unregulated cross-bridge cycling and excessive force production (Beall and Fyrberg, 1991 blue right-pointing triangle).

Figure 3.
Ultrastructure of IFM myofibrils from flies expressing IFI and mutant myosin isoforms. (A) Longitudinal section (left) of wild-type IFM myofibrils at 2 d after eclosion displays easily discernible sarcomeric M-lines and I-, A-, and Z-band structures. ...

Morphological and Functional Effects of Mutant Myosin Activity on Cardiac Muscle

To investigate the effects of depressed or enhanced myosin motor function on cardiac muscle, we surgically exposed, imaged, and tracked beating heart wall movements of semi-intact flies by using direct immersion differential interference contrast optics in conjunction with a high speed digital video camera (see Supplemental Movies 1–3). Visual inspection of the ventral aspect of the heart tubes at 10× magnification revealed obvious differences in mutant cardiac morphology as early as 1 wk after eclosion. Hearts expressing hypoactive D45 myosin seemed considerably dilated compared with wild-type heart tubes (Figure 4A). The D45 heart walls also qualitatively displayed compromised shortening abilities and thus perturbed systolic function. Hyperactive Mhc5 myosin expression induced a narrowing of the heart tubes with obvious restricted regions. These restricted segments seemed unable to adequately relax, and they severely perturbed diastolic function. The restricted region was initially confined to abdominal segment three in several Mhc5 hearts from 1- to 2-wk-old flies. Subsequently, there seemed to be a lateral spread of restriction gradually affecting abdominal segments 2 and 4 of the heart tubes. The overall incidence of the phenotype increased with advancing age such that by 5 wk nearly all hearts showed some sign of restriction and impaired diastolic function.

Figure 4.
Wild-type and mutant heart morphology and function. (A) Top, schematic of the D. melanogaster heart tube, located along the dorsal midline of the abdomen, and the supportive alary muscles (modified from Miller, 1950 blue right-pointing triangle). Hrt, Drosophila heart tube; A1, ...

High-speed videos of contracting heart tubes were used to generate M-mode traces at 3 wk of age (Figure 4B), the age where AIs become increasingly divergent among fly lines (see below; Ocorr et al., 2007 blue right-pointing triangle). These traces qualitatively display the dynamics of cardiac contractions. M-modes from semi-intact 3-wk-old yw and Mhc5 heart preparations show fairly regular contractions. However, the rhythmic beating pattern progressively worsened with age. By 5 wk, both lines exhibited nonrhythmical heart contractile patterns, which included asystoles and fibrillations, reflected by increased AI (see below). In contrast to yw and Mhc5 flies, M-mode records from 3-wk D45 mutant hearts exhibited abundantly more nonrhythmical beating patterns (Figure 4B). Consistent with a substantial AI increase over time (see below) the incidence of arrhythmias became more prevalent by 4 and 5 wk of age in the D45 mutants compared with the other lines.

To confirm the qualitative differences seen among the genotypes, we quantitatively compared the senescent-dependent changes in a number of cardiac parameters from wild-type and mutant hearts. Heart diameters at 1 through 5 wk of age were measured from individual video frames at peak diastolic and systolic time points. All three genotypes exhibited significant decreases in both diastolic and systolic dimensions with age, reflecting a narrowing of the cardiac chamber (Figure 5A). At every age point studied, however, D45 heart walls showed increased mean diastolic and systolic distances, whereas Mhc5 displayed only decreased diastolic diameters relative to wild type. Both wild-type and Mhc5 hearts showed an age-related decrease in fractional shortening, with Mhc5 hearts displaying severely compromised ejection abilities throughout life compared with control hearts (Figure 5B). Fractional shortening for D45 hearts was substantially perturbed at younger ages, relative to that of wild-type hearts; yet, the tubes showed no overall age-related decrease in contractility.

Figure 5.
Analysis of age-dependent changes in physiological parameters of yw (●), D45 (▲), and Mhc5 (■) hearts. Measurements were made on 28–33 hearts from each Drosophila line at 1, 2, 3, 4, and 5 wk of age. The means were jittered ...

We characterized the inherent myogenic contractile dynamics and properties of semi-intact hearts by measuring several cardiac parameters from movies of wild-type and mutant flies from 1 through 5 wk of age. The mean HPs of both yw and Mhc5 flies increased moderately and significantly with age (Figure 5C). In contrast, flies expressing hypoactive D45 myosin exhibited a dramatic and highly significant age-dependent increase in HP compared with age-matched controls. Separating HPs into individual DIs and SIs revealed distinct differences between yw and Mhc5 age-dependent cardiac dynamics (Figure 5, D and E). Over the 5-wk time period, no significant differences were found between DI increases in wild-type and Mhc5 hearts. Hearts from both lines showed similar significant rate increases in SI; however, at all five age points the Mhc5 mean SI was significantly longer than that for yw. Thus, the increase in Mhc5 SI substantially contributed to the prolonged HP seen with advancing age in these mutants. D45 hearts showed significant increases in both DI and SI, a hallmark of failing myocardium. The highly prolonged DI for older D45 hearts was predominately responsible for the progressive and dramatic increase in HP of these mutants.

To explore senescent-dependent increases in heart rhythm irregularities, we compared the AIs of the three lines (Figure 5F). In both wild-type and Mhc5 flies, the AI increased progressively, with no significant differences in rates or overall amounts. The AI for D45 mutant hearts was roughly the same as for control flies at 1 wk of age, and then it increased considerably and significantly compared with control flies, throughout life. Thus, the incidence of arrhythmic cardiac beating patterns increased with age in both wild type and in the mutants, but the extent of increase was much greater in D45 flies.

As discussed below, diminished molecular motor function of D45 myosin in our model system seemed to induce a cardiac phenotype similar to that found in humans with dilated cardiomyopathy (Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle). Enhanced molecular properties of Mhc5 myosin, however, generated pathological hallmarks seemingly analogous to those found in patients suffering from the clinically rare restrictive cardiomyopathy (Kushwaha et al., 1997 blue right-pointing triangle; Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle).

DISCUSSION

Here, we have demonstrated that the myosin transducer is intimately involved with determining the motor's biochemical and mechanical properties and hence the structure and performance of striated muscles. The Drosophila transducer is encoded by constitutively expressed exons so that mutations likely alter the kinetic and mechanical properties in a similar manner in all fly striated muscle motors. Therefore, Drosophila is a valuable model system to explore dysfunction of multiple muscles resulting from enhanced or depressed motor activity. This is particularly beneficial because similar alterations in myosin performance are associated with the development and age-related progression of numerous skeletal and cardiac myopathies found in higher organisms (Sellers, 1999 blue right-pointing triangle; Redowicz, 2002 blue right-pointing triangle; Oldfors et al., 2004 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle; Laing and Nowak, 2005 blue right-pointing triangle; Tardiff, 2005 blue right-pointing triangle; Oldfors, 2007 blue right-pointing triangle).

The Mhc5 mutation maps close to the junction between the HF helix and hypervariable loop 1, a critical element of the transducer. Biochemical and structural studies of wild-type and engineered myosins have shown loop 1 to be involved in kinetic tuning of the motor by influencing a wide range of myosin activities (Kurzawa-Goertz et al., 1998 blue right-pointing triangle; Sweeney et al., 1998 blue right-pointing triangle; Clark et al., 2005 blue right-pointing triangle). The size and flexibility of loop 1 can affect the rate of ATP hydrolysis, product release, actin filament translocation, access to the nucleotide pocket and nucleotide binding affinity (Sweeney et al., 1998 blue right-pointing triangle). Larger and more flexible loops were shown to enhance product release (Sweeney et al., 1998 blue right-pointing triangle). The Mhc5 transducer mutation may straighten the C-terminal segment of the HF helix and increase the length and/or flexibility of loop 1, thereby enhancing the molecular motor's properties.

The D45 transducer mutation is located at the β-bulge/β-strand 7 junction and leads to depressed motor activity. Structural studies showed the transducer's loops and linkers interact with each other and undergo coupled distortions to accommodate the distortion of the central β-sheet that occurs during the ATPase cycle (Coureux et al., 2003 blue right-pointing triangle, 2004 blue right-pointing triangle). The D45 mutation may disrupt vital interactions between distinct transducer elements, disturbing the coordinated distortion of the mutant transducer and the resulting sequential release of products from the nucleotide-binding pocket during force generation (Coureux et al., 2004 blue right-pointing triangle).

The altered motor activity of the Drosophila mutants affected skeletal muscle locomotory function. At young ages, perturbed motor function decreased or eliminated flight ability in D45 or Mhc5 flies, respectively. Both wild-type and D45 flies displayed a characteristic age-dependent reduction in flight ability. Such reductions correlate with declines in glycogen levels and in mitochondrial efficiency and with low myofibrillar protein turnover rates and increased levels of oxidative damage to proteins and lipids (Baker, 1976 blue right-pointing triangle; Magwere et al., 2006 blue right-pointing triangle). Protein oxidation, which leads to inactivation of certain enzyme systems and to structural protein damage, is considered critical for the aging process (Stadtman, 1992 blue right-pointing triangle; Levine and Stadtman, 2001 blue right-pointing triangle; Magwere et al., 2006 blue right-pointing triangle). Thus, extreme physical activity, like flight, poses an inherent, deleterious threat to muscle function. Expression of D45 myosin with depressed motor function may diminish IFM contraction rates and flight capacity, and also metabolic demands, metabolic by-product accumulation, and associated oxidative damage to proteins and lipids of muscle cells. Therefore, the functional decline normally associated with IFM may be decelerated by hypoactive myosin, and in general, motors with diminished molecular properties could provide age-dependent protection in demanding muscles that normally operate at very high contraction rates.

Electron microscopic examination of D45 IFM revealed that depressed myosin activity does not perturb IFM sarcomeric structure. In contrast, although Mhc5 IFM expressing myosin with enhanced molecular function developed normally in the pupa, sarcomeric structure was suddenly destroyed in an apparent use-initiated manner in newly eclosed adults. The ultrastructural phenotype of Mhc5 IFM seems similar to that found in the hypercontracted IFM of hdp2 troponin mutants; both phenotypes have been suggested to result from altered actomyosin interactions (Beall and Fyrberg, 1991 blue right-pointing triangle; Nongthomba et al., 2003 blue right-pointing triangle; Montana and Littleton, 2004 blue right-pointing triangle). hdp2 IFM thin filaments exhibit perturbed contractile regulation by troponin-tropomyosin such that myosin binding sites remain unblocked in both the absence and presence of activating Ca2+ (Cammarato et al., 2004 blue right-pointing triangle). Thus, cross-bridge cycling and force generation would proceed uninhibited and may initiate IFM hypercontraction. Similarly, enhanced kinetic and mechanical properties of Mhc5 myosin may induce excessive cross-bridge cycling and promote cooperative and constitutive activation of thin filaments and force generation. Consequently the contractile system would remain fully activated and, as with hdp2 IFM, which are incapable of relaxing, the fibers could quickly destroy themselves and flight ability would be lost.

Expression of D45 myosin with diminished biomechanical properties produced a Drosophila cardiac phenotype displaying structural and functional characteristics remarkably similar to those found in humans with, and in vertebrate models of, dilated cardiomyopathy (DCM) (Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle; Schmitt et al., 2006 blue right-pointing triangle; Debold et al., 2007 blue right-pointing triangle). DCM is a myocardial disorder characterized by left and/or right ventricular dilation and distended chambers (Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle). Cardiac contractility is depressed, resulting in systolic dysfunction, reduced fractional shortening, and diminished ejection fractions. Affected individuals demonstrate progressive symptoms and gradually develop heart failure, often associated with life-threatening cardiac arrhythmias.

At least 30% of DCM cases are genetic in origin, with more than 15 autosomal dominant missense mutations localized to the β-cardiac Mhc gene (Kamisago et al., 2000 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle; Chang and Potter, 2005 blue right-pointing triangle). Disrupted contractile performance of the diseased myocardium may result from alterations in the mutant myosin's ability to generate force and motion (Schmitt et al., 2006 blue right-pointing triangle; Debold et al., 2007 blue right-pointing triangle). Mouse models engineered with DCM-causing β-cardiac MHC mutations reproduced morphological and functional characteristics consistent with the human phenotype (Schmitt et al., 2006 blue right-pointing triangle). At the molecular level, the mutations depressed ATPase activities, in vitro actin filament sliding velocities, and/or maximal force generating capacity of myosin motors (Schmitt et al., 2006 blue right-pointing triangle; Debold et al., 2007 blue right-pointing triangle). Depressing one or more of these molecular indices of myosin function was considered sufficient to trigger the cascade of events that lead to DCM (Debold et al., 2007 blue right-pointing triangle). Our analysis of D45 Drosophila mutants corroborates this hypothesis. D45 myosin showed substantial decreases in basal and actin-stimulated ATPase rates, and a significant drop in actin sliding velocity. The hearts in turn developed a dilated morphology and functional deficits that worsened with age, consistent with DCM. Thus, the pathological response to depressed motor function seems to be surprisingly similar to that found in higher organisms.

Additional studies have demonstrated a dilatory cardiac response in Drosophila resulting from an N-terminal mutation in the TnI inhibitory troponin subunit of the regulatory complex (Wolf et al., 2006 blue right-pointing triangle). This Drosophila cardiac response is consistent with an N-terminal DCM-causing mutation in human cardiac TnI (cTnI) (Murphy et al., 2004 blue right-pointing triangle; Wolf et al., 2006 blue right-pointing triangle). Drosophila may, therefore, serve as a powerful model for investigating an apparently conserved age-associated cascade of events and cardiac remodeling that occur in response to altered myosin or troponin function that result in specific cardiomyopathies.

In vertebrates, some hypertrophic cardiomyopathy myosin mutations have been shown to increase motor ATPase activities, actin sliding velocities, and/or maximal force generating capacities, suggesting the molecular defects result in a gain of myosin function (Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle; Debold et al., 2007 blue right-pointing triangle). Thus, augmented biophysical properties of individual myosin motors potentially initiate hypertrophic cardiac remodeling. Drosophila heart tubes, however, expressing Mhc5 myosin with enhanced biomechanical properties exhibited a cardiac phenotype displaying structural and functional characteristics remarkably similar to those in human restrictive cardiomyopathy (RCM). RCM is a rare myocardial disorder characterized by cardiac remodeling, decreased myocardial wall elasticity, impaired diastolic ventricular filling, and elevated systemic and pulmonary venous pressures (Kushwaha et al., 1997 blue right-pointing triangle; Fatkin and Graham, 2002 blue right-pointing triangle; Ahmad et al., 2005 blue right-pointing triangle). Cardiac rhythmicity, systolic function, and myocardial wall thickness seem unaffected; however, stroke volume and cardiac output are reduced as a result of diastolic dysfunction and restricted filling. The prognosis for RCM patients is poor in that the majority of them experience progressive deterioration of cardiac function and heart failure with a high incidence of premature mortality.

Six missense cTnI mutations were identified in patients with autosomal dominant RCM (Mogensen et al., 2003 blue right-pointing triangle). Reconstituted troponin complexes with RCM cTnI were unable to properly inhibit actomyosin ATPase (Gomes et al., 2005 blue right-pointing triangle). Replacing endogenous cTnIs with the RCM cTnIs, in skinned cardiac fibers revealed elevated levels of basal force production (Gomes et al., 2005 blue right-pointing triangle; Yumoto et al., 2005 blue right-pointing triangle). Furthermore, relative to wild-type, the RCM cTnI mutations significantly increased the Ca2+ sensitivity of force development in skinned cardiac fibers. The impaired myocardial relaxation seen in RCM patients was proposed to result from an inability of the mutant troponin complex to properly inhibit basal ATPase and force development at low Ca2+ concentrations (Gomes et al., 2005 blue right-pointing triangle). A recent study on membrane intact cardiomyocytes expressing an RCM cTnI mutation indeed resulted in thin filament disinhibition and a Ca2+-independent precontracted basal state (Davis et al., 2007 blue right-pointing triangle).

Our Mhc5 results suggest that increased motor function could also be involved in RCM development. Enhanced kinetic and mechanical activity of this myosin, consistent with inducing IFM hypercontraction, probably promotes excessive cross-bridge cycling in the mutant hearts. Cross-bridge binding physically impedes troponin-tropomyosin regulatory strand movement back to its blocking position on thin filaments and it increases the affinity of troponin for Ca2+, especially in cardiac muscle (Tobacman, 1996 blue right-pointing triangle; Moss et al., 2004 blue right-pointing triangle; Hinken and Solaro, 2007 blue right-pointing triangle). Enhanced myosin cycling may therefore cooperatively promote its own activity by disproportionately increasing the number of available binding sites on actin and by enhancing the Ca2+ sensitivity of the system. Consequently, this could promote the onset of systole and delay relaxation and diastole, prolonging the systolic interval. Mhc5 hearts displayed significantly prolonged systolic intervals, similar to those in children with heart failure secondary to RCM (Friedberg and Silverman, 2006 blue right-pointing triangle). Thus, excessive actin–myosin interactions caused by enhanced motor activity, heightened Ca2+ sensitivity, and/or thin filament disinhibition, possibly initiating high levels of basal tension and extended systolic intervals, may be major determinants of the diastolic dysfunction seen in Mhc5 hearts, and they are likely key components in the pathogenesis of RCM. Myosin mutations have never been associated with the development of RCM. However, because impaired myosin or TnI function induces an apparently evolutionarily conserved dilatory response in the Drosophila model system, myosin mutations that enhance motor properties should also be considered legitimate candidates in the etiology of the clinically rare RCM.

Supplementary Material

[Supplemental Materials]

ACKNOWLEDGMENTS

We thank Douglas Deutschman (San Diego State University) and Douglas M. Swank (Rensselaer Polytechnic Institute) for help with statistical analysis, Chi Lee (San Diego State University) for technical assistance, and Mary C. Reedy (Duke University) for helpful comments and discussions regarding manuscript preparation. This work was supported by a postdoctoral fellowship from the Western States Affiliate of the American Heart Association and National Institutes of Health (NIH) research supplement AR-43396-S1 (to A.C.), NIH grant R01 AR-43396 and NIH grant R01 GM-32443 (to S.I.B.), and NIH grants R01 HL-54732 and P01 AG-15434 (to R.B.).

Abbreviations used:

AI
arrythmicity index
DCM
dilated cardiomyopathy
DI
diastolic interval
FI
flight index
HP
heart period
IFI
indirect flight isoform
IFM
indirect flight muscle
MHC
myosin heavy chain
RCM
restrictive cardiomyopathy
SI
systolic interval
Tn
troponin.

Footnotes

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E07-09-0890) on November 28, 2007.

REFERENCES

  • Ahmad F., Seidman J. G., Seidman C. E. The genetic basis for cardiac remodeling. Annu. Rev. Genomics Hum. Genet. 2005;6:185–216. [PubMed]
  • Baker G. T., 3rd Insect flight muscle: maturation and senescence. Gerontology. 1976;22:334–361. [PubMed]
  • Beall C. J., Fyrberg E. Muscle abnormalities in Drosophila melanogaster heldup mutants are caused by missing or aberrant troponin-I isoforms. J. Cell Biol. 1991;114:941–951. [PMC free article] [PubMed]
  • Bernstein S. I., Mogami K., Donady J. J., Emerson C. P., Jr Drosophila muscle myosin heavy chain encoded by a single gene in a cluster of muscle mutations. Nature. 1983;302:393–397. [PubMed]
  • Brown J. H., Cohen C. Regulation of muscle contraction by tropomyosin and troponin: how structure illuminates function. Adv. Protein Chem. 2005;71:121–159. [PubMed]
  • Cammarato A., Hatch V., Saide J., Craig R., Sparrow J. C., Tobacman L. S., Lehman W. Drosophila muscle regulation characterized by electron microscopy and three-dimensional reconstruction of thin filament mutants. Biophys. J. 2004;86:1618–1624. [PubMed]
  • Chang A. N., Potter J. D. Sarcomeric protein mutations in dilated cardiomyopathy. Heart Fail. Rev. 2005;10:225–235. [PubMed]
  • Clark R., Ansari M. A., Dash S., Geeves M. A., Coluccio L. M. Loop 1 of transducer region in mammalian class I myosin, Myo1b, modulates actin affinity, ATPase activity, and nucleotide access. J. Biol. Chem. 2005;280:30935–30942. [PubMed]
  • Coureux P. D., Sweeney H. L., Houdusse A. Three myosin V structures delineate essential features of chemo-mechanical transduction. EMBO J. 2004;23:4527–4537. [PubMed]
  • Coureux P. D., Wells A. L., Menetrey J., Yengo C. M., Morris C. A., Sweeney H. L., Houdusse A. A structural state of the myosin V motor without bound nucleotide. Nature. 2003;425:419–423. [PubMed]
  • Cripps R. M., Becker K. D., Mardahl M., Kronert W. A., Hodges D., Bernstein S. I. Transformation of Drosophila melanogaster with the wild-type myosin heavy-chain gene: rescue of mutant phenotypes and analysis of defects caused by overexpression. J. Cell Biol. 1994;126:689–699. [PMC free article] [PubMed]
  • Davis J., Wen H., Edwards T., Metzger J. M. Thin filament disinhibition by restrictive cardiomyopathy mutant R193H troponin I induces Ca2+-independent mechanical tone and acute myocyte remodeling. Circ. Res. 2007;100:1494–1502. [PubMed]
  • Debold E. P., Schmitt J. P., Patlak J. B., Beck S. E., Moore J. R., Seidman J. G., Seidman C., Warshaw D. M. Hypertrophic and dilated cardiomyopathy mutations differentially affect the molecular force generation of mouse alpha-cardiac myosin in the laser trap assay. Am. J. Physiol. 2007;293:H284–H291.
  • Drummond D. R., Hennessey E. S., Sparrow J. C. Characterisation of missense mutations in the Act88F gene of Drosophila melanogaster. Mol. Gen. Genet. 1991;226:70–80. [PubMed]
  • Fatkin D., Graham R. M. Molecular mechanisms of inherited cardiomyopathies. Physiol. Rev. 2002;82:945–980. [PubMed]
  • Finch C. E., Ruvkun G. The genetics of aging. Annu. Rev. Genomics Hum. Genet. 2001;2:435–462. [PubMed]
  • Friedberg M. K., Silverman N. H. The systolic to diastolic duration ratio in children with heart failure secondary to restrictive cardiomyopathy. J. Am. Soc. Echocardiogr. 2006;19:1326–1331. [PubMed]
  • Geeves M. A., Fedorov R., Manstein D. J. Molecular mechanism of actomyosin-based motility. Cell Mol. Life Sci. 2005;62:1462–1477. [PubMed]
  • Geeves M. A., Holmes K. C. Structural mechanism of muscle contraction. Annu. Rev. Biochem. 1999;68:687–728. [PubMed]
  • Geeves M. A., Holmes K. C. The molecular mechanism of muscle contraction. Adv. Protein Chem. 2005;71:161–193. [PubMed]
  • George E. L., Ober M. B., Emerson C. P., Jr Functional domains of the Drosophila melanogaster muscle myosin heavy-chain gene are encoded by alternatively spliced exons. Mol. Cell. Biol. 1989;9:2957–2974. [PMC free article] [PubMed]
  • Gomes A. V., Liang J., Potter J. D. Mutations in human cardiac troponin I that are associated with restrictive cardiomyopathy affect basal ATPase activity and the calcium sensitivity of force development. J. Biol. Chem. 2005;280:30909–30915. [PubMed]
  • Gordon A. M., Homsher E., Regnier M. Regulation of contraction in striated muscle. Physiol. Rev. 2000;80:853–924. [PubMed]
  • Hinken A. C., Solaro R. J. A dominant role of cardiac molecular motors in the intrinsic regulation of ventricular ejection and relaxation. Physiology. 2007;22:73–80. [PubMed]
  • Kamisago M., et al. Mutations in sarcomere protein genes as a cause of dilated cardiomyopathy. N. Engl. J. Med. 2000;343:1688–1696. [PubMed]
  • Kronert W. A., Acebes A., Ferrus A., Bernstein S. I. Specific myosin heavy chain mutations suppress troponin I defects in Drosophila muscles. J. Cell Biol. 1999;144:989–1000. [PMC free article] [PubMed]
  • Kurzawa-Goertz S. E., Perreault-Micale C. L., Trybus K. M., Szent-Gyorgyi A. G., Geeves M. A. Loop I can modulate ADP affinity, ATPase activity, and motility of different scallop myosins. Transient kinetic analysis of S1 isoforms. Biochemistry. 1998;37:7517–7525. [PubMed]
  • Kushwaha S. S., Fallon J. T., Fuster V. Restrictive cardiomyopathy. N. Engl. J. Med. 1997;336:267–276. [PubMed]
  • Laing N. G., Nowak K. J. When contractile proteins go bad: the sarcomere and skeletal muscle disease. Bioessays. 2005;27:809–822. [PubMed]
  • Levine R. L., Stadtman E. R. Oxidative modification of proteins during aging. Exp. Gerontol. 2001;36:1495–1502. [PubMed]
  • Magwere T., Pamplona R., Miwa S., Martinez-Diaz P., Portero-Otin M., Brand M. D., Partridge L. Flight activity, mortality rates, and lipoxidative damage in Drosophila. J. Gerontol. A Biol. Sci. Med. Sci. 2006;61:136–145. [PubMed]
  • Margossian S. S., Lowey S. Structural and contractile proteins. In: Cunningham L. W., Frederiksen D. W., editors. Methods in Enzymology. Vol. 85. New York: Academic Press; 1982. pp. 55–70.
  • McKillop D. F., Geeves M. A. Regulation of the interaction between actin and myosin subfragment 1, evidence for three states of the thin filament. Biophys. J. 1993;65:693–701. [PubMed]
  • Miller A. The internal anatomy and histology of the imago of Drosophila melanogaster. In: Demerec M., editor. Biology of Drosophila. New York: Wiley; 1950. pp. 420–534.
  • Mogensen J., Kubo T., Duque M., Uribe W., Shaw A., Murphy R., Gimeno J. R., Elliott P., McKenna W. J. Idiopathic restrictive cardiomyopathy is part of the clinical expression of cardiac troponin I mutations. J. Clin. Invest. 2003;111:209–216. [PMC free article] [PubMed]
  • Montana E. S., Littleton J. T. Characterization of a hypercontraction-induced myopathy in Drosophila caused by mutations in Mhc. J. Cell Biol. 2004;164:1045–1054. [PMC free article] [PubMed]
  • Moss R. L., Razumova M., Fitzsimons D. P. Myosin crossbridge activation of cardiac thin filaments: implications for myocardial function in health and disease. Circ. Res. 2004;94:1290–1300. [PubMed]
  • Murphy R. T., Mogensen J., Shaw A., Kubo T., Hughes S., McKenna W. J. Novel mutation in cardiac troponin I in recessive idiopathic dilated cardiomyopathy. Lancet. 2004;363:371–372. [PubMed]
  • Nongthomba U., Cummins M., Clark S., Vigoreaux J. O., Sparrow J. C. Suppression of muscle hypercontraction by mutations in the myosin heavy chain gene of Drosophila melanogaster. Genetics. 2003;164:209–222. [PubMed]
  • Ocorr K., Reeves N. L., Wessells R. J., Fink M., Chen H. S., Akasaka T., Yasuda S., Metzger J. M., Giles W., Posakony J. W., Bodmer R. KCNQ potassium channel mutations cause cardiac arrhythmias in Drosophila that mimic the effects of aging. Proc. Natl. Acad. Sci. USA. 2007;104:3943–3948. [PubMed]
  • Oldfors A. Hereditary myosin myopathies. Neuromuscul. Disord. 2007;17:355–367. [PubMed]
  • Oldfors A., Tajsharghi H., Darin N., Lindberg C. Myopathies associated with myosin heavy chain mutations. Acta Myol. 2004;23:90–96. [PubMed]
  • Pardee J. D., Spudich J. A. Structural and contractile proteins. In: Cunningham L. W., Frederiksen D. W., editors. Methods in Enzymology. Vol. 85. New York: Academic Press; 1982. pp. 164–179.
  • Parkes T. L., Hilliker A. J., Phillips J. P. Motorneurons, reactive oxygen, and life span in Drosophila. Neurobiol. Aging. 1999;20:531–535. [PubMed]
  • Paternostro G., Vignola C., Bartsch D. U., Omens J. H., McCulloch A. D., Reed J. C. Age-associated cardiac dysfunction in Drosophila melanogaster. Circ. Res. 2001;88:1053–1058. [PubMed]
  • Poole K. J., Lorenz M., Evans G., Rosenbaum G., Pirani A., Craig R., Tobacman L. S., Lehman W., Holmes K. C. A comparison of muscle thin filament models obtained from electron microscopy reconstructions and low-angle X-ray fibre diagrams from non-overlap muscle. J. Struct. Biol. 2006;155:273–284. [PubMed]
  • Redowicz M. J. Myosins and pathology: genetics and biology. Acta Biochim. Pol. 2002;49:789–804. [PubMed]
  • Root D. D., Wang K. Calmodulin-sensitive interaction of human nebulin fragments with actin and myosin. Biochemistry. 1994;33:12581–12591. [PubMed]
  • Rozek C. E., Davidson N. Drosophila has one myosin heavy-chain gene with three developmentally regulated transcripts. Cell. 1983;32:23–34. [PubMed]
  • Schmitt J. P., et al. Cardiac myosin missense mutations cause dilated cardiomyopathy in mouse models and depress molecular motor function. Proc. Natl. Acad. Sci. USA. 2006;103:14525–14530. [PubMed]
  • Sellers J. R. Oxford; New York: Oxford University Press; 1999. Myosins.
  • Stadtman E. R. Protein oxidation and aging. Science. 1992;257:1220–1224. [PubMed]
  • Suggs J. A., Cammarato A., Kronert W. A., Nikkhoy M., Dambacher C. M., Megighian A., Bernstein S. I. Alternative S2 hinge regions of the myosin rod differentially affect muscle function, myofibril dimensions and myosin tail length. J. Mol. Biol. 2007;367:1312–1329. [PMC free article] [PubMed]
  • Swank D. M., Bartoo M. L., Knowles A. F., Iliffe C., Bernstein S. I., Molloy J. E., Sparrow J. C. Alternative exon-encoded regions of Drosophila myosin heavy chain modulate ATPase rates and actin sliding velocity. J. Biol. Chem. 2001;276:15117–15124. [PubMed]
  • Swank D. M., Knowles A. F., Kronert W. A., Suggs J. A., Morrill G. E., Nikkhoy M., Manipon G. G., Bernstein S. I. Variable N-terminal regions of muscle myosin heavy chain modulate ATPase rate and actin sliding velocity. J. Biol. Chem. 2003;278:17475–17482. [PubMed]
  • Sweeney H. L., Rosenfeld S. S., Brown F., Faust L., Smith J., Xing J., Stein L. A., Sellers J. R. Kinetic tuning of myosin via a flexible loop adjacent to the nucleotide binding pocket. J. Biol. Chem. 1998;273:6262–6270. [PubMed]
  • Tardiff J. C. Sarcomeric proteins and familial hypertrophic cardiomyopathy: linking mutations in structural proteins to complex cardiovascular phenotypes. Heart Fail. Rev. 2005;10:237–248. [PubMed]
  • Tatar M., Bartke A., Antebi A. The endocrine regulation of aging by insulin-like signals. Science. 2003;299:1346–1351. [PubMed]
  • Tobacman L. S. Thin filament-mediated regulation of cardiac contraction. Annu. Rev. Physiol. 1996;58:447–481. [PubMed]
  • Vibert P., Craig R., Lehman W. Steric-model for activation of muscle thin filaments. J. Mol. Biol. 1997;266:8–14. [PubMed]
  • Wessells R. J., Bodmer R. Age-related cardiac deterioration: insights from Drosophila. Front. Biosci. 2007;12:39–48. [PubMed]
  • Wolf M. J., Amrein H., Izatt J. A., Choma M. A., Reedy M. C., Rockman H. A. Drosophila as a model for the identification of genes causing adult human heart disease. Proc. Natl. Acad. Sci. USA. 2006;103:1394–1399. [PubMed]
  • Yumoto F., et al. Drastic Ca2+ sensitization of myofilament associated with a small structural change in troponin I in inherited restrictive cardiomyopathy. Biochem. Biophys. Res. Commun. 2005;338:1519–1526. [PubMed]

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