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Vol. 19, Issue 2, 553-562, February 2008
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*Department of Biology and Heart Institute, San Diego State University, San Diego, CA 92182-4614; Development and Aging Program, Burnham Institute for Medical Research, La Jolla, CA 92037; and Department of Chemistry and Biochemistry, San Diego State University, San Diego, CA 92182-1030
Submitted September 12, 2007;
Revised November 6, 2007;
Accepted November 16, 2007
Monitoring Editor: Thomas Pollard
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2005; Geeves et al., 2005). 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; Redowicz, 2002; Oldfors et al., 2004; Chang and Potter, 2005; Laing and Nowak, 2005; Tardiff, 2005; Oldfors, 2007). 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). 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). 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; Sweeney et al., 1998; Clark et al., 2005). 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).
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; Brown and Cohen, 2005). 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; Vibert et al., 1997; Poole et al., 2006). 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; Gordon et al., 2000; Moss et al., 2004). 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; Rozek and Davidson, 1983; George et al., 1989). 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; Magwere et al., 2006) and cardiac muscles (Paternostro et al., 2001; Wolf et al., 2006; Ocorr et al., 2007). 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; Finch and Ruvkun, 2001; Tatar et al., 2003; Wessells and Bodmer, 2007). 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; Montana and Littleton, 2004). 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). 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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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) 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) for determining myosin concentrations.
ATPase Assays
Myosin ATPase activities were determined using [
-32P]ATP. Ca2+ ATPase was measured as described previously (Swank et al., 2001). Actin-activated ATPase was determined using chicken skeletal muscle actin (Swank et al., 2003). G-actin was isolated from acetone powder of chicken skeletal muscle according to Pardee and Spudich (1982). 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), 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), by using the modifications described in Swank et al. (2001). 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) and Suggs et al. (2007). 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). 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). M-modes were generated using a MatLab (MathWorks, Natick, MA)-based image analysis program (Ocorr et al., 2007). 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.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). 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). 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, 2004). 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). 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.
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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).
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). 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.
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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; Ahmad et al., 2005; Chang and Potter, 2005). 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; Fatkin and Graham, 2002; Ahmad et al., 2005).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Redowicz, 2002; Oldfors et al., 2004; Chang and Potter, 2005; Laing and Nowak, 2005; Tardiff, 2005; Oldfors, 2007).
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; Sweeney et al., 1998; Clark et al., 2005). 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). Larger and more flexible loops were shown to enhance product release (Sweeney et al., 1998). 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, 2004). 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).
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; Magwere et al., 2006). Protein oxidation, which leads to inactivation of certain enzyme systems and to structural protein damage, is considered critical for the aging process (Stadtman, 1992; Levine and Stadtman, 2001; Magwere et al., 2006). 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; Nongthomba et al., 2003; Montana and Littleton, 2004). 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). 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; Ahmad et al., 2005; Chang and Potter, 2005; Schmitt et al., 2006; Debold et al., 2007). DCM is a myocardial disorder characterized by left and/or right ventricular dilation and distended chambers (Fatkin and Graham, 2002; Ahmad et al., 2005; Chang and Potter, 2005). 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; Ahmad et al., 2005; Chang and Potter, 2005). 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; Debold et al., 2007). Mouse models engineered with DCM-causing β-cardiac MHC mutations reproduced morphological and functional characteristics consistent with the human phenotype (Schmitt et al., 2006). 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; Debold et al., 2007). 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). 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). This Drosophila cardiac response is consistent with an N-terminal DCM-causing mutation in human cardiac TnI (cTnI) (Murphy et al., 2004; Wolf et al., 2006). 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; Ahmad et al., 2005; Debold et al., 2007). 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; Fatkin and Graham, 2002; Ahmad et al., 2005). 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). Reconstituted troponin complexes with RCM cTnI were unable to properly inhibit actomyosin ATPase (Gomes et al., 2005). Replacing endogenous cTnIs with the RCM cTnIs, in skinned cardiac fibers revealed elevated levels of basal force production (Gomes et al., 2005; Yumoto et al., 2005). 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). 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).
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; Moss et al., 2004; Hinken and Solaro, 2007). 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). 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.
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ACKNOWLEDGMENTS |
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Footnotes |
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Present address: The Scripps Research Institute, Kellogg School of Science and Technology, 10550 N. Torrey Pines Rd., TPC-19, La Jolla, CA 92037. 
Address correspondence to: Sanford I. Bernstein (sbernst{at}sciences.sdsu.edu) or Karen Ocorr (kocorr{at}burnham.org)
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.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), D45 (
) and Mhc5 (
) myosin molecules. ATPase results are represented with data fitting lines. Vmax values obtained in actin-stimulated Mg2+ ATPase assays by using D45 myosin are reduced to nearly a third of the values obtained for the IFI, whereas Vmax values for the Mhc5 isoform are slightly reduced compared with that of the IFI. Km values do not differ (see 
