Severe Mechanical Dysfunction in Pharyngeal Muscle from Adult mdx Mice PIERRE ATTAL, FRANCINE LAMBERT, SYLVAIN MARCHAND-ADAM, SERGE BOBIN, JEAN-CLAUDE POURNY, DENIS CHEMLA, YVES LECARPENTIER, and CATHERINE COIRAULT Service d’Oto-Rhyno-Laryngologie et de Chirurgie Cervico-Faciale, Hôpital de Bicêtre; INSERM U451-Loa-ENSTA-Ecole Polytechnique, Palaiseau Cedex; and Service de Physiologie Cardio-vasculaire et Respiratoire, UER Paris XI, Hôpital de Bicêtre, Assistance Publique–Hôpitaux de Paris, Le Kremlin-Bicêtre, France
The mdx mouse is a widely used animal model of human muscular dystrophy. Although diaphragm muscle exhibits severe muscle weakness throughout the life of the animal, the limb muscle function of mdx mice spontaneously recovers by 6 mo of age. Pharyngeal dilator muscles such as sternohyoid (SH) contribute to upper airway patency during breathing. We hypothesized that SH muscle function was impaired in 6-mo-old mdx mice. Mechanical properties and myosin heavy chain (MHC) composition were investigated in isolated SH from 6-mo-old control (C, n ⫽ 10) and mdx (n ⫽ 10) mice. As compared with C, peak tetanic tension (Pmax) and maximum shortening velocity were 50% and 16% lower in mdx mice (p ⬍ 0.001 and p ⬍ 0.05, respectively). Peak mechanical power was lower in mdx than in C (19.0 ⫾ 3.2 versus 57.4 ⫾ 5.1 mW g⫺1, p ⬍ 0.001). Both C and mdx SH were composed exclusively of fast myosin isoforms. As compared with C, mdx SH presented a higher proportion of IIX-MHC and a reduction in IIB-MHC (each p ⬍ 0.001). In conclusion, our results demonstrated severe SH muscle dysfunction in 6-mo-old mdx mice, that is, at a time when limb muscle function has recovered. Thus, SH muscle of the mdx mouse may be an excellent muscle for studying Duchenne muscular dystrophy.
The genetic similarities between mdx mice and Duchenne muscular dystrophy (DMD) have made the mdx mouse an extremely attractive model for the study of human muscular dystrophy. In both cases, a point mutation in the dystrophin gene results in a lack of the membrane-associated protein, dystrophin (1–3). In mdx mice, the expression of muscular dystrophy differs in diaphragm versus limb skeletal muscles. After an early period of muscle degeneration (2–4 wk) in which muscle weakness is manifest, limb skeletal muscles of mdx mice show a remission of their disease conditions and benign progression of the pathology (14–16 wk) (4). In contrast, the diaphragm muscle of mdx mice undergoes a progressive degeneration similar to that occurring in muscles of humans with DMD (5), leading to severe muscle atrophy and weakness (5, 6). Abnormal pharyngeal muscle function is frequently seen in DMD (7–9). To the best of our knowledge, the mechanical properties of the pharyngeal muscles have not yet been documented in mdx mice. The aim of our study was to determine whether intrinsic sternohyoid (SH) muscle function was impaired in 6-mo-old mdx mice. Pharyngeal muscles contribute to upper airway patency during breathing in awake and sleeping subjects (10). Pharyngeal dilator muscles such as SH exhibit a phasic activity during inspiration. Two hypotheses were tested: (1) that decreased intrinsic SH muscle function is
present in 6-mo-old dystrophic mice; (2) that changes in the mechanical properties are associated with changes in the expression of myosin isoform.
METHODS Experimental Protocol Animal care was in keeping with the recommendations of the Helsinki declaration. Experiments were conducted on 20 strips of sternohyoid (SH) muscle from 6-mo-old male mdx mice (n ⫽ 10) and agematched control mice (C57BL/10ScSn, n ⫽ 10) obtained from the Jackson Laboratory (Bar Harbor, ME). The mice were anesthetized with pentobarbital sodium (40 mg/kg intraperitoneally). Sternohyoid strips were removed via a sagittal cervicotomy after separation of the submaxillary glands. First, the hyoid bone was identified and its superior muscular attachments were cut. The hyoid bone was then cut medially and the two sternohyoid muscles were separated along the middle line as far as the inferior end of the muscles, requiring a sternotomy. A strip of SH was obtained by cutting one SH muscle laterally. The muscular strip was kept attached to a piece of bone at each end (hyoidal and sternal ends). Each muscle strip was vertically suspended in a bath containing the following Krebs–Henseleit solution (in mM): 118 NaCl, 4.7 KCl, 1.2 MgSO4, 1.1 KH2PO4, 24 NaHCO3, 2.5 CaCl2, and 4.5 glucose. The solution was bubbled with 95% O2–5% CO2 and maintained at 26⬚ C (pH 7.4). The sternal end of the SH was held in a stationary clip at the bottom of the bath and the hyoidal end was held in a spring clip attached to an electromagnetic lever system. Experiments were carried out at L0, that is, the initial resting length corresponding to the apex of the initial length-active tension curve. Muscle strips were stimulated by means of two platinum electrodes in tetanic conditions as follows: train duration, 250 ms; train frequency, 6/min. Maximum isometric tension was generally achieved at a stimulation frequency of 100 Hz. At the end of the experiment, the cross-sectional area (in square millimeters) was calculated from the ratio of fresh muscle weight to muscle length at L0, assuming a muscle density of 1.056. The characteristics of the studied muscle strips were as follows: L0; 6.9 ⫾ 0.4 and 6.2 ⫾ 0.4 mm in control and mdx sternohyoid, respectively (NS).
The Electromagnetic System The load applied to the muscle was determined by means of a servocontrolled current through the coil of an electromagnet. Force amplitude could be measured between 0 and 140 mN, with an error of less than 1%. Muscle shortening displaced the lever, thereby modulating the light intensity received by a photoelectric transducer. The linearity of the system ranged from 0 to 5 mm of muscle shortening, and the error was less than 0.5% of full-scale deflection. All analyses were based on digital records obtained with a microcomputer. Two signals, tension and length, were recorded. Software for calculating all the parameters was developed in our laboratory.
Mechanical Parameters (Received in original form May 18, 1999 and in revised form December 2, 1999) Correspondence and requests for reprints should be addressed to Dr. C. Coirault, INSERM 451-LOA-Ecole Polytechnique, Batterie de l’Yvette, 91761 Palaiseau Cedex, France. E-mail:
[email protected] Am J Respir Crit Care Med Vol 162. pp 278–281, 2000 Internet address: www.atsjournals.org
Eight to 10 contractions were performed, in which the load was regularly incremented from zero load up to isometry. The peak isometric tension, that is, peak force normalized per cross-sectional area, was measured from the fully isometric contraction (Pmax, in mN mm⫺2). Maximum unloaded shortening velocity (VZL, in L0/s) was measured from the contraction abruptly clamped to zero load just after stimulus (11).
Attal, Lambert, Marchand-Adam, et al.: Pharyngeal Muscle in mdx Mouse The peak velocity (V) of afterloaded contractions was plotted against the isotonic load level normalized per cross-sectional area (P), obtained by successive load increments . from zero load up to the isometric tension. Mechanical power ( W ) was calculated over the whole load continuum. We thus determined peak mechanical power . . ( W max, expressed in mW g⫺1). To ensure that comparisons of W val. ues were made at similar levels of relative load, each experimental W versus P relationship was fitted using a five-order polynomial function. The equations of the fitted curves were entered into a computer using the TransERA HTBasic Advanced Math Library (TransERA . Corporation, Provo, UT), which calculated W at fixed relative tension (i.e., P ⫽ 10, 20, 30, 40, 50, 60, 70, 80, 90, 100% Pmax). Finally, the tension–velocity relationship was also studied. The experimental P–V relationship was fitted according to Hill’s equation (12): (P ⫹ a) (V ⫹ b) ⫽ (cPmax ⫹ a)b, where ⫺a and ⫺b are the asymptotes of the hyperbola as determined by multilinear regression and the least-squares method, and cPmax is the calculated peak isometric tension at V ⫽ 0. The G curvature of the hyperbolic tension–velocity relationship, which is linked to the myothermal economy and cross-bridge kinetics, was determined according to the formula G ⫽ cPmax/a (13, 14).
Myosin Electrophoresis Preparations of crude myosin were obtained from sternohyoid muscle, as previously described (15). Electrophoresis was performed in a BioRad Mini-Protean II Dual Slab Cell electrophoresis system for 29 h at 4⬚ C and 70 V (constant voltage). Myosin heavy chains (MHC) were separated in dissociating conditions with sodium dodecyl sulfate–polyacrylamide (SDS–PAGE) minigel electrophoresis (16, 17). Stacking gel was composed of 4% acrylamide (2.67% bis-acrylamide), 70 mmol Tris (pH 6.8), 30% glycerol, 4 mmol ethylenediaminetetraacetic acid (EDTA), and 0.1% SDS. The composition of the separating gel was 8% acrylamide (1% bis-acrylamide), 200 mmol Tris (pH 8.8), 100 mmol glycine, and 0.4% SDS. Separate upper and lower running buffers were used. The upper running buffer consisted of 100 mmol Tris (base), 150 mmol glycine, 11.5 mmol 2-mercaptoethanol, and 0.1% SDS. The lower running buffer consisted of 50 mmol Tris (base), 75 mmol glycine, and 0.05% SDS. Both buffers were prepared shortly before use and cooled at 4⬚ C. Gels were stained with 0.2% Coomassie blue, 50% ethanol, and 10% acetic acid, and destained with 5% ethanol and 5% acetic acid. The different MHC isoforms were quantified by one-dimensional densitometry (GS-690; BioRad, Hercules, CA). The area of each peak was used to determine the amount of each isoform. Data were expressed as percentages of the area of each peak over the sum of the areas of all peaks. Identification of the specific myosin isoform bands was based on bands from costal diaphragm and on previous studies (17).
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Figure 1. Mechanical parameters in control (C) and mdx mouse sternohyoid. (Left panel) Peak isometric force normalized per cross-sectional area (Pmax). (Right panel ) Maximum unloaded shortening velocity (VZL). Values are means ⫾ SEM (n ⫽ 10 in each group); *p⬍ 0.05, **p⬍ 0.001 as compared with controls. L0 is the initial muscle length at which active tension is maximum.
C and mdx, respectively). Force–velocity relationships in C and mdx mice are shown in Figure 3. The force–velocity relationship was more curved in mdx than in control SH, with a significantly higher G curvature in mdx than in C (p ⬍ 0.001) (Figure 3). Myosin Isoform Composition
The electrophoretic separation of the different MHC isoforms present in normal and mdx mouse SH is shown in Figure 4. Both control and mdx SH were composed exclusively of type IIX and IIB MHC isoforms. As compared with the control group, mdx SH contained a greater proportion of type IIX MHC (64 ⫾ 2 versus 16 ⫾ 1%) (p ⬍ 0.001), whereas the proportion of type IIB MHC was lower (36 ⫾ 2 versus 84 ⫾ 1%, p ⬍ 0.001). None of the muscles demonstrated type I or type IIA MHC isoforms.
DISCUSSION This is the first study to report the mechanical function of a pharyngeal muscle from the mdx mouse, namely the sternohyoid muscle (SH). We demonstrated that 6-mo-old mdx mice exhibited intrinsic SH muscle dysfunction, as attested by sig-
Statistical Analysis Data are expressed as means ⫾ SEM. After analysis of variance (ANOVA), comparisons between groups were performed using Student’s unpaired t test. All comparisons were two-tailed and a p value of ⬍ 0.05 was considered significant.
RESULTS Contractile Performance of SH
Mechanical parameters of SH muscles are presented in Figure 1. Peak isometric tension (Pmax) was markedly depressed in mdx mice compared with age-matched controls (p ⬍ 0.001). The percentage difference between mdx and C was 艐 50%. In addition, maximum shortening velocity (VZL) was 艐 16% lower in the mdx mouse group than in controls (p ⬍ 0.05). Figure 2 depicts the mechanical power in both C and mdx mice. In both groups, the mechanical power versus tension relationship was bell shaped, indicating a marked influence of loading conditions on mechanical power. The peak mechani· cal power (W max) was higher in C than in mdx (57.4 ⫾ 5.1 versus 19.0 ⫾ 3.2 mW g⫺1, p ⬍ 0.001). On the other hand, the relative load at which Wmax occurred (PW· max) did not significantly differ between groups (34 ⫾ 1 versus 32 ⫾ 2% Pmax in
· Figure 2. Interpolated mechanical power (interpolated W ) versus load (P) (expressed as a percentage of Pmax) relationship in sternohyoid (SH) from control (C, n ⫽ 10) and mdx mice · (mdx, n ⫽ 10). Pmax, maximum isometric tension. Interpolated W was calculated using a five-order· polynomial function (see METHODS) to ensure that comparisons of W were made at similar levels of relative load. Means ⫾ SE are indicated.
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Figure 4. Typical electrophoretic profiles of myosin heavy chain (MHC) in control and mdx mouse sternohyoid muscles (SH). Identification of the specific myosin isoform bands was based on diaphragm (Dia) bands from control mice. Figure 3. (Left panel) Superimposed representative tension–velocity relationships in sternohyoid from control (C) and mdx mice. (Right panel) G curvature of the tension–velocity relationship in sternohyoid from C (n ⫽ 10) and mdx mice (n ⫽ 10). Means ⫾ SE are indicated. *p ⬍ 0.05 as compared with controls.
nificant decreases in muscle strength, muscle velocity, and mechanical power. Sternohyoid muscle weakness was associated with changes in myosin isoform pattern, characterized by a shift from IIB to IIX-MHC isoforms. Animal Model
The mdx mouse lacks the same sarcolemmal protein missing from skeletal muscles in human DMD (18, 19). The genetic similarities between mdx and DMD have made the mdx mouse an extremely attractive model for the study of human muscular dystrophy. Numerous studies, however, have revealed that mdx mice do not suffer from a progressive skeletal muscle disease but rather experience an acute episode of muscle degeneration at 2–4 wk of age, followed by muscle regeneration that is completed by at least 12–16 wk of age (20, 21). In particular, the limb muscles, and especially the fast-twitch ones, exhibit a spontaneous functional recovery despite the lack of dystrophin (4). The one exception identified to date is the diaphragm, which is unique in mdx mice in showing ongoing necrosis and progressive fibrosis throughout the life of the animal (22) as well as abnormal mechanical properties (5, 6, 23, 24). Intrinsic SH Muscle Function
As compared with control mice, our results showed that SH muscle strength in 6-mo-old mdx mice was reduced by almost 50%. The reduction in SH tension approximated that observed in diaphragm from age-matched mdx mice (5, 6, 24). In various muscle groups from the mdx mice, the decline in muscle force runs parallel to the progression of muscular necrosis and/or fibrosis (5, 21). The myopathic process may interfere with muscle fiber contractile properties. In keeping with this hypothesis, we recently demonstrated that in diaphragm muscle the unitary force produced per cross-bridge was significantly lower in mdx mice than in control mice (24). However, in diaphragm muscle, the difference in unitary force produced per cross-bridge between control and myopathic mice is about 5%, whereas fat and connective tissue infiltration represent about 50% of the total muscle area (5, 22). Thus, our results suggested that SH muscle is involved in the myopathic degenerative process in adult mdx mice, that is, at a stage at which normal limb muscle function has been restored (21). Further histological studies are needed to confirm this hypothesis. Differences in fiber-type composition may also account, at least in part, for differences in maximum muscle tension found in dystrophic muscles. The amount of force generated by slow myosin isoforms has been reported to be higher than that produced by fast myosin isoforms, whereas no significant differ-
ences in force-generating capacities have been reported between the different fast myosin isoforms (25). However, given that both mdx and C SH muscles were composed exclusively of fast myosin isoforms, it is unlikely that relative changes in fast myosin isoforms between groups could account for the reduced SH muscle strength. As compared with controls, 6-mo-old mdx SH exhibited a relative increase in IIX-MHC and a corresponding decrease in IIB-MHC (Figure 4). Previous studies have reported that type IIX fibers have a lower maximum shortening velocity and lower actomyosin ATPase activity than IIB fibers (26, 27). Thus, the fast myosin isoform shift reported in mdx SH muscle corresponded to a transition toward a slower phenotype, a finding consistent with previous studies in mdx mice (6). Moreover, changes in myosin isoforms in mdx SH probably accounted for reduced maximum shortening velocity associated with reduced power output (Figure 1) (28). The ability to generate mechanical (i.e., external) power is a key muscle property that determines, at least in part, physiological activity. Mdx SH differed from controls in terms of peak mechanical power and myothermal economy. In both groups, mechanical power was strongly dependent on loading conditions and peak mechanical power occurred at similar relative load levels. However, peak mechanical power was onethird as high in mdx than in control SH muscle (Figure 2). In contrast, the G curvature of the force–velocity relationship was higher in mdx SH than in controls (Figure 3), thus suggesting more favorable economical behavior of mdx as compared with control SH (12, 14). Possible Implications
The precise mechanisms by which 5- to 6-mo-old mdx muscles recover or fail to recover adequate function in the absence of dystrophin have not yet been established. In synergy with the diaphragm, pharyngeal muscles contribute to upper airway patency during breathing in awake and sleeping subjects. Pharyngeal dilator muscles such as SH exhibit a phasic activity during inspiration. One possible explanation for the difference between SH and diaphragm on the one hand and limb skeletal muscles on the other hand is that chronic rhythmical activity contributes to the more pronounced deleterious changes in diaphragm muscle (5) and also SH. Numerous protocols are in progress for treating muscular dystrophy, including gene therapy (29, 30). One difficulty in precisely evaluating the functional benefits of these treatments is that limb muscles spontaneously recover in mdx mice. We suggest that SH from mdx mice may provide an appropriate model for evaluating the functional impacts of treatment muscle-wasting diseases. In conclusion, our results documented sternohyoid muscular dysfunction in 6-mo-old mdx mice, that is, at a time where limb skeletal muscles have recovered. Thus, SH muscle of mdx mice may provide a satisfactory model for the study of myopathic disease.
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