INTRODUCTION

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Keywords: in vitro motility assays; mdx mice; crossbridge; myosin

Duchenne muscular dystrophy (DMD) and the mdx mouse are characterized by the lack of dystrophin, a protein that connects the subsarcolemmal cytoskeleton to the extracellular matrix (1, 2). Progressive degeneration and reduction in the amount of contractile tissue largely account for muscle wasting in DMD and mdx diaphragm (3). However, the exact pathogenesis of muscular dystrophy is still a subject of debate. It has been proposed that lack of dystrophin alters the integrity of the sarcolemma, leading to focal breakdowns of the sarcolemma (2, 4). This results in cell necrosis and progressive replacement of contractile muscle fibers by connective tissue. Increased sarcolemmal Ca²⁺ influx via abnormally functioning mechanosensitive channels (5) or Ca²⁺ leak channels (6, 7) have also been reported in mdx muscles. This may in turn be responsible for greater resting intracellular Ca²⁺ in mdx muscle fibers and activation of Ca²⁺-dependent proteases (7) leading eventually to muscle necrosis (10, 11).

Additional abnormalities may contribute to the impaired muscle performance in dystrophic mouse muscle. The sarcoplasmic reticulum Ca²⁺ pump has been reported to be functionally altered in mdx muscle (10, 12). In addition, recent studies have suggested that functional alterations of the contractile apparatus may also play a role in impaired diaphragm muscle performance (4, 13, 14). Using Huxley's mathematical model of muscle contraction (15, 16), we have previously reported impaired crossbridge interactions in the diaphragm muscle from the mdx mouse compared with control mice (14). Moreover, this later study suggests that changes in the kinetics of myosin molecular motors cannot be explained solely by changes in myosin heavy chain isoforms (14). However, the interpretation of crossbridge kinetics in whole diaphragm muscle is limited by a number of factors that may modulate actomyosin interactions, such as delayed sarcoplasmic Ca²⁺ movements (10, 12) and/or decreased compliance of mdx diaphragm (3, 17). Purified actin and myosin molecules in in vitro motility assays are an effective alternative way of studying the kinetics of actomyosin interactions without the confounding effects related to intact multicellular preparations (18).

In the present study, in vitro motility assays were used to analyze the functional properties of myosin from mdx diaphragm and limb muscles. In contrast to diaphragm, which shows ongoing necrosis and progressive fibrosis throughout the life of the mdx mouse as well as abnormal mechanical properties (4, 14, 19), limb muscles exhibit spontaneous functional recovery despite the lack of dystrophin (3, 11, 17, 19). Thus, by comparing in vitro motility of diaphragm and limb muscle myosins it was possible to determine whether potential abnormal myosin function was a consequence of the lack of dystrophin. Myosin isoform composition (MHC) was also determined. We tested the hypothesis that the myosin molecule was functionally altered in the mdx muscle. Our results showed that myosin extracted from mdx diaphragm muscle moved actin filaments at a lower velocity than that from control diaphragm, despite nonsignificant changes in the slow-to-fast ratio in diaphragm myosin isoforms. This suggests that the myosin molecule is functionally altered in dystrophic mouse diaphragm.

	METHODS

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Purification of Contractile Proteins

Myosin preparations were obtained from 9-mo-old male mdx mice (n = 12) and age-matched control mice (n = 12) (Jackson Laboratory, Bar Harbor, ME). Care of the animals conformed to the Helsinki convention. The age of 9 mo was chosen because at this age diaphragm of mdx animals is dramatically impaired, whereas limb muscle has almost recovered (3, 11, 17, 19). After anesthesia with pentobarbital sodium (40 mg/kg, intraperitoneally), the costal diaphragm and then the semitendinous muscle from each hindlimb were excised. All procedures were performed at 4° C. Muscles were minced and homogenized in 3 volumes of Guba-Straub solution (18). After centrifugation, the supernatant was precipitated in 10 volumes of ice-cold distilled water with 5 mmol dithiothreitol (DTT). After 30 min, the myosin was collected by centrifugation, dissolved in 3 mol KCl, and again precipitated in 10 volumes of ice-cold distilled water with 5 mmol DTT. After 15 min, the myosin was collected by centrifugation and resuspended in myosin buffer. The purity of the final myosin solution was checked by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Fresh myosin, purified less than 72 h before, was used for the experiments. Actin was prepared from rabbit back muscle (20) and fluorescently labeled with tetramethylrhodamine-phalloidin (Molecular Probes).

In Vitro Motility Assays

In vitro motility assays were performed as previously described (21, 22). The protein samples and buffer solutions were infused into the microscope flow cell at 90-s intervals in the following order. First, the myosin sample was diluted to 40 µg/ml in a high ionic strength buffer and applied to the flow cell. Unbound myosin was then washed out with high-salt buffer bovine serum albumin (BSA, 0.5 mg/ml). Motility assay buffer (AB) with 0.5 mg/ml BSA was then infused into the flow cell. Unlabeled F-actin filaments were used to block nonfunctional myosin molecules. Then, fluorescent actin filaments in AB were added at a concentration of 70 ng/ml. Unbound actin was washed out by AB with an oxygen-scavenger enzyme system. Motility of actin filaments was initiated by adding 2 mmol ATP. Recordings were made at controlled room temperature (22° C).

The movement of actin filaments was observed under a Zeiss epifluorescence microscope (100/1.30 objective) with an intensified camera (Zaï 7550500, Copenhagen, Denmark) and recorded on a video tape. Analysis of filament velocities was performed using N.J Carter's freeware RETRAC program (http://mc11.mcri.uk/retrac).

Myosin Electrophoresis

Electrophoresis was performed in a Bio-Rad Mini-Protean II Dual Slab Cell electrophoresis system. The MHC composition was determined by SDS-polyacrylamide minigel electrophoresis as previously described (14, 23, 24). The acrylamide concentrations were 4% in the stacking gel and 8% in the separating gel. The gels were run for 32 h at 4° C and then stained with Coomassie blue. The relative amounts of the different MHC were quantified by densitometry (14).

Statistical Analysis

Data are expressed as mean ± SD. The mean velocity and SD were calculated for each filament. A two-tailed unpaired Student's t test was used to determine whether difference between group means is significant. A value of p < 0.05 was considered significant.

	RESULTS

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Velocity of Actin Sliding over Myosin

Frequency histograms of velocities of actin filament sliding over myosins from control and mdx mice are shown in Figures 1 and 2. Motility velocity driven by myosin extracted from control diaphragm (1.9 ± 0.1 µm · s¹, n = 153) was significantly faster than in the case of myosin from mdx diaphragm (1.2 ± 0.1 µm · s¹, n = 153, p < 0.001). Conversely, there was no significant difference between motility velocity driven by myosin from control and mdx hindlimb muscles (2.4 ± 0.1 µm · s¹ versus 2.5 ± 0.1 µm · s¹, n = 155 filaments in each group). The motility speed with myosin from control and mdx hindlimb was significantly faster than actin motility speeds propelled by diaphragm myosin (each p < 0.001).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Distribution of velocities of actin filament sliding over myosin from mdx and control mouse diaphragms. Individual frame-to-frame velocities from tracked filaments are plotted in a frequency histogram. Each histogram contains data from 153 filaments taken from six separate assays. For each assay, myosin was obtained from two diaphragm muscles to obtain a sufficient amount of tissues.

View larger version (16K): [in this window] [in a new window]   Figure 2.   Distribution of velocities of actin filament sliding over myosin from mdx and control mouse semitendinous muscles. Individual frame-to-frame velocities from tracked filaments are plotted in a frequency histogram. Each histogram contains data from 153 filaments taken from six separate assays. For each assay, myosin was obtained from two leg muscles to obtain a sufficient amount of tissue.

Myosin Isoform Composition

MHCs isoforms from diaphragm and semitendinous muscle were classified as type I, IIA, IIX, or IIB according to the order of migration (Figure 3). No significant differences were observed in control and mdx diaphragms as regards slow MHC (6 ± 2% versus 7 ± 3%) or the fast-to-slow MHC ratio. As compared with control diaphragm, mdx diaphragm contained a greater amount of type IIA MHC (60 ± 5% versus 35 ± 3%, p < 0.001), whereas the proportion of type IIX MHC was significantly lower (27 ± 6% versus 48 ± 4, p < 0.0001). Type IIB myosin isoforms, which represented 11 ± 4% MHC in control diaphragm, represented only 6 ± 2% MHC in mdx diaphragm (p < 0.01). In semitendinous muscle, control mice expressed almost exclusively type IIB MHC. The majority of the semitendinous muscles from mdx mice (n = 6 of 10) also expressed almost exclusively type IIB MHC (Figure 3). However, type IIX myosin isoforms were also present in 4 of 10 semitendinous muscles from mdx mice and represented 3 ± 4% of the total myosin isoform.

View larger version (43K): [in this window] [in a new window]   Figure 3.   Typical electrophoretic profiles of myosin heavy chain in normal (N) and mdx mouse diaphragm (DIA) and semitendinous (SQ) muscles. Identification of the specific myosin isoform bands was based on diaphragm bands from control rats (C).

	DISCUSSION

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This is the first study to report the velocity of actin filament sliding over surfaces coated with myosin molecules from the dystrophic mouse. Our results showed that the velocity of actin filament sliding over myosin from mdx mouse diaphragm was slower compared with myosin from control diaphragm, despite nonsignificant changes in the slow-to-fast ratio in diaphragm myosin isoforms. No significant difference was observed between control and mdx myosin-based velocity in hindlimb muscle. These results are in good agreement with the proposal that the myosin molecular motor is functionally altered in 6-mo-old dystrophic mouse diaphragm (14).

In isolated muscle, we have previously reported impaired crossbridge interactions in the diaphragm muscle from the mdx mouse (14). However, mechanical studies on isolated muscle are limited in that they cannot unambiguously determine whether differences in the actomyosin interaction cycle arise from differences in the molecular motor itself, from variations in metabolic or ionic conditions inside the fiber, or from variations in the regulatory proteins of contraction. In vitro motility assays ensure that variations in filament velocities between control and mdx muscle are due to intrinsic variations in myosin molecules (18, 21, 25).

Potential mechanisms that may contribute to the reduced filament velocity in mdx diaphragm myosin may involve myosin isoform diversity (26), variations in mechanical constraints opposing the sliding movement (27), or pathological changes in the enzymatic or mechanical properties of myosin (28). In normal skeletal muscle and for a given experimental temperature, the observed filament velocity is largely dependent on MHC isoform expression (25) and decreases as the proportion of slow MHC isoform increases (26, 27). The relative MHC composition for control mouse diaphragm was consistent with that previously reported (24, 31). Compared with controls, 9-mo-old mdx diaphragm exhibited no significant change in the proportion of slow myosin isoforms. Therefore, the reduced myosin-based velocity cannot be attributed to a change in the fast-to-slow ratio in myosin isoforms. Among fast myosins, both MHC and the myosin light chain (MLC) isoforms help modulate the velocity of actin translocation, although in a rather complex interplay (for a review see [26]). In studies performed on a mixture of different fast myosin isoforms, no significant differences in maximum velocity between type IIA and type IIX fibers have been identified (32). Moreover, the influence of changes in the MLC isoforms is much lower in fibers containing IIX and IIA MHC than in fibers containing IIB MHC (35). Taken as a whole, these findings strongly suggest that the shift from IIX to IIA-MHC isoforms observed in mdx diaphragm myosin (Figure 3; 14) is an unlikely explanation for the reduced sliding velocity.

Alternatively, noncycling myosins are known to decrease the sliding velocity generated by the cycling myosins, possibly by producing a resistive drag on filament sliding (27). This point was addressed in our study by adding unlabeled F-actin filaments in the motility assay. The fact that these filaments block nonfunctional myosin molecules (21, 36) argues against noncycling myosin-induced reduction in motility velocity in mdx diaphragm.

Other regulatory mechanisms have been proposed to modulate velocity of movement in in vitro motility assays. Recent studies have provided evidence that structural changes in the myosin molecule itself can slow filament motility independent of myosin isoform diversity (28). Myosin extracted from aged muscle fibers moves actin filaments at a lower speed than that from young adult animals despite nonsignificant variations in MHC or MLC isoforms (28). Furthermore, an electron paramagnetic resonance study has confirmed structural changes in the myosin head that can contribute to alterations in myosin function in aged muscle fibers (30). It can be concluded from these studies that myosin diversity is not restricted to changes in myosin isoforms but may also involve subtle changes in amino acids within the myosin head, at least in old age (28). No structural data are yet available for myosin from dystrophic mouse diaphragm. However, in line with these findings, it may be speculated that structural modifications also occur in myosin from mdx diaphragm, which would help explain the reduced motility velocity in mdx compared with control mice.

Potential structural changes that lead to reduced motility velocity driven by diaphragm myosin remain to be elucidated. However, it is interesting to note that within one species, structural differences between MHC isoforms involve relatively large regions of the myosin head, the nonidentical amino acid being as high as 20% (26). Two flexible loops, located near the nucleotide- and the actin-binding sites of the myosin head, have been proposed as major determinants of the kinetics properties of myosin (37, 38). Modification of a limited number of amino acids within these loops was found to modify ATPase activity and actin filament translation in vitro (38). It has been proposed that in old age, posttranslational modifications of the myosin structure may account for altered myosin function (28, 30). Moreover, it has been suggested that these changes are related to specific loss of cysteine residues in skeletal muscle myosin (30). Dystrophic diaphragm muscle cells contain elevated levels of intracellular Ca²⁺, which is believed to activate calpains, ubiquitous calcium-dependent cysteine proteases (6, 8, 9). It is thus possible that enhanced calpain activation within dystrophic cells modifies the structure of the myosin head, thereby reducing the velocity of actin translocation in dystrophic diaphragm. This possibility needs to be investigated further.

We found higher sliding velocity with myosin from semitendinosus, a fast muscle type composed mainly of type IIB MHC, than with that from control diaphragm. These finding are in accordance with the higher motility velocity reported in type IIB myosin compared with type IIA MHC (36). Moreover, no significant difference in myosin-based velocity was observed between control and mdx limb muscles. These results suggest that abnormal myosin function is not an inherent consequence of a lack of dystrophin. Although it is difficult to differentiate primary pathogenic from secondary events in chronic muscular dystrophy, a comparative analysis of diaphragm and limb myosin function may be a step toward discriminating between these processes. It is noteworthy that after 4 mo, mdx limb muscle regains almost complete function and structure, whereas the diaphragm undergoes progressive muscle fiber degeneration (3, 17). Increased intracellular Ca²⁺ (11) and calpain activation (9) are both observed in fibers showing evidence of degeneration but not in morphologically normal fibers from the same mdx muscle. By analogy, we suggest that abnormal myosin function may occur only in fibers that are already compromised by the underlying degenerative process.

Care must be taken when transferring results obtained from in vitro motility assays to a complex in vivo conditions. However, the abnormal myosin function found in mdx diaphragm may have clinical implications. In both diaphragm (3, 4, 17) and pharyngeal mdx muscle (39), the decline in muscle function is progressive, as seen in patients with DMD, and human dystrophic fibers undergo repeated episodes of degeneration and regeneration. Although in human and mouse dystrophic muscles, most of the reduction in force-generating capacity is a result of reduction in contractile tissue, our results suggest that myosin dysfunction may further aggravate respiratory muscle weakness. Because muscle shortening properties of the diaphragm in vivo partially determine respiratory work, abnormal actomyosin velocity may affect respiratory muscle function. In addition, our study provides new insights into the pathophysiology of muscular dystrophy that may have potential implications for the treatment of DMD patients.

In conclusion, our results showed reduced maximum velocity of actin filament sliding over myosin from mdx diaphragm muscle, suggesting alterations in the crossbridge mechanics themselves. Given that motility velocity was not altered in mdx limb muscle, our results suggest that dysfunction in the diaphragm myosin may be a consequence of muscle cell damage rather than a primary effect of the lack of dystrophin per se.