INTRODUCTION

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MEROSIN IS AN EXTRACELLULAR protein that is believed to transmit muscle force between the sarcolemma and collagen fibers (17). Merosin functions in cell attachment by providing pathways between the collagen and the transmembrane proteins (20), and this occurs through two pathways. The first pathway involves merosin binding to -dystroglycan of the dystrophin-glycoprotein complex. An alternate pathway involves merosin binding to ₇₁-integrin of the integrin complex. These pathways allow for interactions between collagen and neighboring muscle fibers via merosin. Absence of merosin could create a disruption in both pathways, thereby eliminating the communication between the collagen and the transmembrane proteins.

Merosin deficiency causes congenital muscular dystrophy (18), and this affects skeletal muscles as well as peripheral nerves (22, 24, 26), so peripheral neuropathy is found in a high percentage of affected persons (15). Merosin-deficient congenital muscular dystrophy in humans is characterized by several symptoms, including severe hypotonia, multiple severe contractures, generalized weakness and atrophy, markedly delayed motor development, and frequent severe respiratory insufficiency (9). The disease results from an abnormality or absence of one of the three merosin subunits, the ₂-subunit, coded for at chromosome 6 at the 6q2 position (29).

The dy/dy mouse lacks merosin in skeletal muscle and peripheral nerves and has severely defective basement membranes in the muscle (25, 32). The dy/dy mouse shares many of the hallmarks of the human form of congenital muscular dystrophy and therefore is a good mouse model for this disease (32, 33). The dy/dy mouse develops muscle weakness at ~3 wk of age (12), which progressively worsens, and by 8 wk of age the histological changes in muscles are significant. In the dy/dy mouse, the muscle initially appears normal, with normal fiber-type differentiation up to 2 wk of age, when the necrotic process starts. After 1 mo of age, there is marked variation in fiber size with necrotic and regenerating fibers (30). Interstitial fibrosis becomes evident as the disease progresses.

The role of the extracellular matrix protein merosin in force transmission in skeletal muscles is unknown. In this study, we investigated the mechanical properties of the diaphragm and biceps femoris hindlimb muscles in the dy/dy mouse. We tested the hypothesis that merosin deficiency alters muscle compliance, viscoelastic properties, and muscle contractile force production. To test this hypothesis, we determined the axial and transverse passive length-tension relationships in these muscles. We also examined the viscoelastic properties of the diaphragm in response to constant mechanical stretch in either the fiber or transverse to fiber direction. In addition, we determined the isometric contractile properties of the diaphragm during uniaxial and biaxial loading.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Measurements of passive mechanical properties. We used eight C57BL/6J-Lama2dy-2J mice (weight: 13.4 ± 1.1 g; age: 55.7 ± 7.6 days) and eight normal C57BL/6J mice (weight: 20.5 ± 1.8 g; age: 50.7 ± 2.1 days) obtained from the Jackson Laboratory for our experiments. All animal studies were approved by the Baylor College of Medicine Advisory Committee for Animal Use and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. It is important to note that the control mice used were either wild type or heterozygotes. At this age, the effect of merosin deficiency is clearly observed in the C57BL/6J-Lama2dy-2J through severe weakness of either one or both hindlimbs (Fig. 1).

View larger version (118K): [in this window] [in a new window]   Fig. 1.   Left: 5-mo-old merosin-deficient C57BL/6J-Lama2dy-2J mouse. Right: 5-mo-old normal C57BL/6J mouse. The merosin-deficient mouse has reached terminal stages of the condition, suffering complete loss of locomotor control in the hindlimbs and having both hindlimbs appearing to extend at an abnormal angle from the body. The hindlimb muscles are the only muscles that are paralyzed in the dy/dy mice, and this is possibly because the sciatic nerve is also affected in these muscles.

Muscle mechanical testing. After excision, either the diaphragm or biceps femoris was then placed in an in vitro biaxial muscle testing apparatus. This apparatus has two perpendicular axes, each driven by a micrometer. Four small identical alligator clamps, one for each side of the muscle, were used to hold the muscle during passive muscle lengthening and shortening maneuvers. The diaphragm muscle was secured by fixing one clamp on the central tendon and the opposing clamp on the muscle-tendonous junction at the chest wall insertion with the rib cage intact. The biceps femoris muscle was secured by fixing opposing clamps on the muscle near the muscle-tendonous junction. The muscle was mounted so that the position of these markers during stretching and shortening could be viewed with a black-and-white CCTV-type camera and recorded on a videocassette. Two force transducers (World Precision Instruments, model FORT 100, ±50 g, differential bridge type) were used to measure passive muscle force. Each transducer has a resolution of 0.01% full scale, a sensitivity of 3 µV · V¹ · g¹, and a hysteresis of 0.1% full scale. One transducer for each axis was used to measure the forces applied to the muscle during passive lengthening and forces released during passive muscle shortening. The forces were then amplified and recorded with the use of LABVIEW software version 6.0. The recorded video was digitally captured with a video capture card by using a frame grabber at a sampling rate of 2 Hz. The markers on the captured video were then digitized with Image Tool V2.0 (www.ddsdx.uthscsa.edu), and the precise marker position was determined on Cartesian coordinate axes, assuming that all markers lie within the same plane.

Two-dimensional surface strains. The mechanical strains were calculated according to Boriek et al. (7) with some modifications. The region enclosed by the markers was divided into triangles, with the markers forming the apexes of the triangles. The three points that define a triangle determine a plane. The coordinates of these points in the plane at the reference position were denoted as x_i and y_i (i = 1, 2, 3). The displacements in the local coordinate system of the markers from their reference positions to their positions at a deformed state are denoted by u_i and v_i. These are assumed to be a linear function of position in the plane of the triangle. For example, the following equation

(1)

with the measured values of the displacements and position of any three adjacent markers substituted for u_i, x_i, and y_i, provided a set of three equations for three coefficients, a₁, a₂, and a₃. Similarly, the data provided the information required to determine the coefficients in the following equation, where a₄, a₅, and a₆ are constants

(2)

Strains along the muscle fibers (x) and strains transverse to the fibers (y) that occurred during passive lengthening and shortening of the muscles were computed according to the following equations (4)

(3)

(4)

where u and v denote the marker's displacement in the x (along fibers) and y (transverse to fibers) directions, respectively. The values of a₂, a₃, and so forth were substituted in Eqs. 3 and 4 to obtain u/x, u/y, and so forth. The values of x and y reflect fractional changes in the direction along the muscle fibers and transverse to the fibers, respectively. Calculations of strains were made relative to the unstressed length of the muscle. Unstressed length was defined as the length where the muscle was placed in the tissue bath in the absence of applied mechanical forces. We computed the extensibility ratio,  = 1 + , where  is the mechanical strain in either the fiber or transverse to the fiber direction.

Measurements and modeling of viscoelastic properties. After producing the length-tension curves, we measured stress-relaxation curves in the same merosin-deficient and control mice. The behavior of the living tissue is sensitive to the history of muscle deformation; therefore, before uniaxial stress-relaxation maneuvers, we preconditioned the muscle by loading the muscle tissue to L_o for five cycles. In the axial direction, the muscle was stretched to L_o and kept at that length. In the transverse fiber direction, the muscle was stretched by a passive force equivalent to that applied in the axial direction and kept at that length. The muscle tension was allowed to relax symptotically until it essentially reached a plateau. Forces were collected at 10 Hz by using a force transducer.

Measurement of isometric contractile properties of the diaphragm. Diaphragms from eight normal C57BL/6J mice (weight: 18.6 ± 1.9 g; age: 63.0 ± 4.0 days) and nine merosin-deficient C57BL/6J-Lama2dy-2J mice (weight: 16.9 ± 3.8 g; age: 81.6 ± 17.5 days) were used. After animals were anesthetized, we excised the left hemidiaphragms and immediately placed them in a muscle bath with continuously circulating oxygenated 95% O₂-5% CO₂ Krebs-Ringer solution. The muscle was clamped on both the central tendon and ribs, with one clamp being connected to a Cooper Instruments LQB 630 force transducer. After the muscle was positioned between two stainless steel mesh electrodes, it was stimulated by a Grass S88 stimulator. L_o was determined by twitch responses (0.1-ms stimulus duration, supramaximal voltage), and we tetanically stimulated the diaphragm muscle at 100 Hz preceded by twitches (supramaximal voltage, 0.5-ms pulses, tetanic train duration of 500 ms, 120-s recovery). The muscle was then clamped transverse to the long axis of the muscle fibers, with one clamp attached to a World Precision Instruments FORT250 force transducer and the other clamp attached to a force carriage. Tetanic stimulation sequences were repeated three times in the presence of either 1- or 2-g passive forces that were applied in the direction transverse to muscle fibers. These measurements were collected at room temperature (25°), which minimized temperature-dependent deterioration of the preparation. The contractile force data were collected at a sample rate of 300 Hz with the use of a data acquisition board (model Lab-PC-1200/AI, National Instruments) and LabVIEW software (version 4.0). The force data were stored in an ASCII file for postanalysis.

Immunolabelings. Muscles were frozen in isopentane precooled in liquid nitrogen. Transverse sections (5 µm thick) were preincubated for 20 min in phosphate-buffered saline (pH 7.2) containing 5% albumin. Serial sections were incubated overnight at 4°C with anti-collagen IV.

Statistical analysis. Differences between groups were assessed by ANOVA with use of the SAS Procedure "Mixed" Program (23). The model was a two-factor fixed or random effects model for two groups of mice (dy/dy null vs. controls) and two treatments (passive mechanics: uniaxial stretch along the muscle fibers vs. uniaxial stretch transverse to fibers; active mechanics: uniaxial loading vs. biaxial loading). A P value of 0.05 was chosen as the acceptable level of significance throughout the analysis of all data.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Passive stiffness is increased in muscles from the dy/dy mice. Data in Fig. 2 show representative length-tension curves for the dy/dy and control mice. Both loading (lengthening) and unloading (shortening) curves are shown. The data demonstrate that, during lengthening, there is a slow and continuous increase in tension over the range of imposed strains. Both dy/dy and controls exhibited hysteresis; that is, at the same tension, the muscle exhibited lower mechanical strain on lengthening than on shortening. It appears that hysteresis is smaller in the dy/dy muscles. Furthermore, the axial length-tension curves of the normal diaphragm shifts to the right relative to the transverse length-tension curve. This suggests that the muscle has greater extensibility in the direction of muscle fibers than in the transverse direction to the fibers. The length-tension curves of the dy/dy diaphragm exhibit similar behavior. Extensibility ratios () for all the dy/dy and control mice were computed at a tension of 5 g/cm. In the direction along the fibers (AF) of the diaphragm muscle,  is smaller in the dy/dy mice compared with control mice (control:  = 1.35; dy/dy:  = 1.21). In the direction transverse to muscle fibers (TF),  is smaller in the dy/dy mice than in the control mice (control:  = 1.21; dy/dy:  = 1.17). At a tension of 5 g/cm, the tensile strain in the direction of the fibers is 21 and 35% for dy/dy and control mice, respectively, yielding a compliance ratio of 0.90. A compliance ratio that is less than one implies that the muscle is less compliant compared with its counterpart. The tensile strain at the same level of tension transverse to the muscle fibers is 17 and 21%, respectively, yielding a compliance ratio of 0.97. These data suggest that muscles lacking merosin are less extensible and less compliant than those in normal mice.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Length-tension relationships of the diaphragm muscle for representative dy/dy and age-matched control mice. The diaphragm muscles were passively lengthened and passively shortened in the directions along and transverse to the muscle fibers.  and , Lengthening and shortening curves in the direction along the muscle fibers for control and dy/dy mice, respectively. + and * , Lengthening and shortening curves in the direction transverse to the fibers for control and dy/dy mice, respectively. The curves for the dy/dy mice in the direction along or transverse to the muscle fibers are shifted to the left compared with the controls. At a tension of 5 g/cm, the tensile strain in the direction of the fibers is 21 and 35% for dy/dy and control mice, respectively, yielding a compliance ratio of 0.90. The tensile strain at the same level of tension transverse to the muscle fibers is 17 and 21%, respectively, yielding a compliance ratio of 0.97. These data suggest that muscles lacking merosin are less extensible and less compliant than normal muscles.

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Length-tension relationships of the biceps femoris for dy/dy and age-matched control mice. The muscles of the biceps femoris were passively lengthened and passively shortened in the directions along and transverse to the muscle fibers.  and , Lengthening and shortening curves in the direction along the muscle fibers for control and dy/dy mice, respectively. + and * , Lengthening and shortening curves in the direction transverse to the fibers for control and dy/dy mice, respectively. The curves for the dy/dy mice in either axially or transversely loaded muscle fibers are shifted to the left compared with controls. At a tension of 5 g/cm, the tensile strain in the direction of the fibers is 11 and 46% for dy/dy and control mice, respectively, yielding a compliance ratio of 0.76. The tensile strain at the same level of tension transverse to the muscle fibers is 17 and 23%, respectively, yielding a compliance ratio of 0.95. These data suggest that muscles lacking merosin are less extensible and less compliant than the normal muscles.

View larger version (31K): [in this window] [in a new window]   Fig. 4.   Uniaxial and biaxial muscle passive length-tension relationships for dy/dy and age-matched control mice.  and +, Axial length-tension curves for control and dy/dy mice, respectively.  and * , Biaxial length-tension curves for control and dy/dy mice, respectively. For both the dy/dy and controls, the axial length-tension curves are shifted to the right compared with those during biaxial loadings. The shift is much more pronounced in the normal mouse than in the dy/dy mouse. That is, the effect of transverse stress on the axial length-tension curve is less pronounced in the merosin-deficient mouse. These data suggest that stiffening effect due to biaxial loading is reduced in the merosin-deficient mice.

Merosin contributes to muscle viscoelasticity. A linear spring instantaneously produces a deformation proportional to the load, whereas a dashpot produces a velocity proportional to the load at any instant. Furthermore, a dashpot represents a damping term proportional to velocity. The dashpot relaxation time is the length of time necessary for the dashpot, or shock-absorbing quality of muscle, to be essentially relaxed, that is, at the completely relaxed point the muscle is characterized as elastic. The dashpot relaxation times () for merosin-deficient diaphragms compared with controls are shown in Fig. 5. Because is equivalent to the ratio of the dashpot to the linear spring in series (₁µ₁), an increase in indicates that the dashpot component has a greater effect than the linear spring in series for any given loading condition. was significantly greater in merosin-deficient diaphragms compared with normal muscle. This was true during biaxial loading and during uniaxial loading (P < 0.05). These data suggest that the absence of merosin alters the viscoelastic properties of the diaphragm muscle. In contrast to normal muscles, differences between uniaxial loading and biaxial loading vanished. That is, the effect of transverse load on the values of E_R is only apparent in normal muscles but not in merosin-deficient muscles. These data suggest that the absence of merosin alters the viscoelastic properties of the diaphragm muscle by increasing the relaxation time, and this effect is more pronounced during transverse loading as well as during biaxial loading.

View larger version (14K): [in this window] [in a new window]   Fig. 5.   Dashpot relaxation time () for merosin-deficient mouse diaphragms compared with controls. Muscle was passively lengthened in the direction either along the fiber or transverse to the fiber. The muscle was then held constant, and forces were recorded until a plateau was reached. Because is equivalent to the ratio of the dashpot to the linear spring in series (1µ1), an increase in indicates that the dashpot component has a greater effect than the linear spring in series for any given loading condition. The was significantly greater in merosin-deficient mice compared with control mice in the direction transverse to the muscle fibers (P < 0.05). For both merosin-deficient and control diaphragms, was greater along the fibers compared with biaxial loading conditions (P < 0.05).

View larger version (22K): [in this window] [in a new window]   Fig. 6.   A: relaxed elastic modulus (ER) values for merosin-deficient mice diaphragms compared with controls. During stress relaxation in the direction of the muscle fibers, ER was essentially the same for both the mutant and control mice. During either uniaxial transverse loading conditions or biaxial loading, however, muscles from the mutant mice exhibited significantly lower values of ER than those of controls (P < 0.005). These data suggest that muscle relaxed stiffness is smaller in transverse plane of merosin-deficient diaphragms compared with controls. B: stress-relaxation curves fitted to raw data for merosin-deficient and control diaphragms. The 2 curves represent the stress-relaxation data in the transverse fiber direction.  and , Raw data. Fitted data are represented by dashed lines. ER values were smaller in merosin-deficient diaphragms compared with controls (P < 0.002). These data show that merosin contributes to viscous response of the diaphragm muscle.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Maximal muscle stress production (N/cm2) in control and merosin-deficient diaphragm muscles under uniaxial and biaxial loading conditions at frequency of stimulation of 100 Hz. Data were obtained from 7 normal C57BL/6J mice (weight: 18.6 ± 1.9 g; age: 63.0 ± 4.0 days) and 8 merosin-deficient C57BL/6J-Lama2dy-2J mice (weight: 16.9 ± 3.8 g; age: 81.6 ± 17.5 days). Under uniaxial loading, muscle stress production is depressed in the absence of merosin (P < 0.05), whereas there was no significant difference in contractile stress between mutant and normal mice during biaxial loading. Transverse passive force of either 1 or 2 g increases contractile muscle stress production in both the mutant and control mice (P < 0.05). These data demonstrate that merosin-deficient muscles have altered contractile response.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   Merosin deficiency causes decreased hindlimb muscle force production. Mean ± SE muscle tetanic stresses in normal and desmin-null biceps femoris under uniaxial and biaxial loading are shown. Data at 100-Hz stimulation were obtained from 7 normal C57BL/6J mice (weight: 18.6 ± 1.9 g; age: 63.0 ± 4.0 days) and 8 merosin-deficient C57BL/6J-Lama2dy-2J mice (weight: 16.9 ± 3.8 g; age: 81.6 ± 17.5 days). Under either uniaxial or biaxial loading, muscle stress production is decreased in the absence of merosin (ANOVA: P < 0.01). Unlike the normal diaphragm muscle, the effect of biaxial loading on normal hindlimb muscle is not significantly different from those muscles that are loaded uniaxially.

View larger version (90K): [in this window] [in a new window]   Fig. 9.   The amount of extracellular matrix in hindlimb muscles from a 2-mo-old mutant dy/dy mouse and an aged-matched control mouse was estimated by indirect immunofluorescence with antibodies to type IV collagen. Fibrosis is present in the dy/dy mice, and little is present in wild-type mice.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

The mechanical properties of diaphragm muscle are dependent on the properties of the cells and the interconnections with the extracellular matrix. Merosin is considered one of the very essential extracellular elements that form this junction between the extracellular matrix and the muscle cytoskeleton. To date, no previous studies have investigated the mechanical properties of skeletal muscles in the dy/dy mouse. In this study, we measured length-tension curves, the viscous response, and maximal tetanic stress in the diaphragm from the dy/dy mouse, a mouse model of merosin deficiency. Our results demonstrate that muscle extensibility and compliance are decreased in the diaphragm and biceps femoris muscles of the dy/dy mouse. Furthermore, compared with controls, E_R is greater in the deficient diaphragm muscle. Additionally, maximal tetanic force is depressed in the dy/dy mouse during uniaxial and biaxial loading.

Our data show that the normal diaphragm exhibits characteristics of viscoelastic response to mechanical loading. This is consistent with classic data on muscle viscoelasticity (14). Furthermore, recent studies from our laboratory (2, 5, 6) demonstrated that passive mechanical properties of diaphragm muscles in the AF direction are different from those in the TF direction. These studies showed that the diaphragm is less compliant in the TF direction compared with that in the AF direction. Most recently, our laboratory (21) has found two distinct signaling pathways of force transmission in the AF and TF directions. For both control and merosin-deficient mice, length tension curves in the TF direction are shifted to the left compared with those in the AF direction, indicating greater muscle extensibility along the muscle fibers. However, independent of the direction of passive stretch, merosin-deficient muscles demonstrate a leftward shift of the length-tension curves compared with controls. This suggests that merosin-deficient muscles are less extensible and that a greater tension is required to passively lengthen muscles from the mutant mice to the same muscle length as that of the normal mice. The length-tension curves of the biceps femoris, shown in Fig. 3, demonstrate that for the normal muscles the axial length-tension curves are shifted to the right of that in the transverse direction. In the dy/dy mouse, however, differences in length-tension curves between those in the axial direction and those in the transverse direction appear to be negligible. This indicates that the muscle sheet of the biceps femoris in the absence of merosin is nearly isotropic. That is, mechanical properties may be independent of the direction of mechanical stretch in merosin-deficient muscles. The data also demonstrate that the merosin-deficient muscle is essentially inextensible in either the fiber or the transverse to fiber direction. This is consistent with loss of muscle function in the hindlimb muscles of the dy/dy mice at this age. This is also consistent with upregulation of collagen IV in these muscles at that age.

In vivo, the diaphragm muscle unlike most other skeletal muscles of the hindlimb and forelimbs experiences loads in the AF direction as well as in the TF direction. Any muscle sheet that is loaded uniaxially in the AF direction would have different length-tension relationship than when the sheet is also loaded in the TF direction. Therefore, this complicates the use of uniaxial in vitro properties to analyze in vivo behavior of the diaphragm muscle. Conducting in vitro physiological experiments under biaxial loading of the diaphragm muscle is therefore critical to the understanding of diaphragm function. Therefore, the effect of biaxial loading or stresses applied in the two perpendicular directions of the muscle sheet of the diaphragm was investigated. The results show that length-tension curves during biaxial loading are much stiffer compared with axially loaded muscles. This is consistent with previous data on length-tension curves of the intact, pressurized loaded muscles and excised uniaxially loaded muscle sheets of the rat diaphragms (5). Furthermore, the data show that the merosin-deficient diaphragm is less extensible compared with controls when experiencing biaxial loading.

The dashpot relaxation time () is the time necessary for the dashpot or shock-absorbing quality of the muscle to be essentially relaxed; that is, at this point the muscle is characterized as perfectly elastic. The was significantly greater in the transverse plane of the muscle in merosin-deficient mice compared with control mice. That is, in the absence of merosin, the diaphragm muscle appears to require more time to reach the perfect elastic state when loaded in the direction orthogonal to the long axis of the muscle fibers. E_R provides a quantitative value for the viscous response of the muscle, where an E_R of 1.0 signifies a perfect elastic behavior. The results show that, compared with controls, the merosin-deficient diaphragm has a smaller E_R value in the transverse plane. Both and E_R data strongly suggest that the merosin-deficient diaphragm is more viscous in the transverse plane compared with controls. Therefore, these data suggest that, at least in the transverse plane, merosin decreases the damping capacity of the muscle.

The effect of age and breeding constraints are important to the understanding of the significance of the results of this study. The contractile force capacity of merosin-deficient muscles is most likely decreased with age, and this is because merosin deficiency causes progressive muscle degeneration. It was impossible to conduct our experiments on these mice in the prenecrotic stage because these mice develop muscle necrosis shortly after birth. Merosin-deficient mice are infertile, and, as such, heterozygotes must be mated to generate the merosin-deficient mouse. However, genotyping studies on these mice are inconclusive, and, as a result, distinguishing between controls and knockouts must be made on the basis of phenotype. Merosin-deficient mice can be easily phenotypically distinguished from normal mice on the basis of gait, where these mice display an obvious paralysis of the hindlimbs at ~40 days old. The mice reported in the present study are of that age (~6-7 wk); therefore, muscles from these mice were tested after the onset of the disease. In merosin-deficient mice, there is an upregulation of ₇₁-integrin in skeletal muscles (19, 31), suggesting that integrin engagement is needed to substitute for some of the loss of mechanical integrity of the cells in the absence of merosin.

In earlier studies, we and our colleagues (3, 4, 8) investigated muscle fiber architecture and mechanism(s) of force transmission in the diaphragm of the dog. These studies demonstrated that muscle fiber architecture is complex and mostly discontinuous. That is, there are fibers that either span the entire length of the muscle or terminate by tapering before reaching either or both attachments. We reasoned that, when the diaphragm is submaximally activated, as during normal breathing and maximal inspiratory efforts, muscle force could be transmitted to the cell membrane and to the extracellular intramuscular connective tissues by shear linkage, presumably via structural transmembrane proteins. The results of our present study are consistent with merosin being a possible candidate protein that transmits muscle force from the cell membrane to collagen fibers. During submaximal activation, merosin could also transmit muscle force from the active muscle fibers of the diaphragm to the adjacent passive muscle fibers, and the mechanism of force transmission is most likely in shear.

Increased collagen and fibrosis are general features of muscular dystrophy, and increased muscle stiffness has generally been attributed to an increased proportion of collagen (1, 13, 16, 27, 28). However, some studies have demonstrated lack of correlation between the increased amount of collagen and passive stiffness. In particular, in soleus muscles of the rat, the changes in muscle stiffness were not associated with increased collagen with aging. In addition, Coirault et al. (11) found that muscle stiffness was decreased in the diaphragm muscles from Syrian hamster, whereas surface area of collagen was increased in these animals. On the basis of these reports, muscle passive stiffness is not always correlated with increase in the proportion of collagen. Therefore, the increased stiffness in the merosin-deficient muscles is not necessarily entirely due to the increase in collagen seen in the dy/dy mice at ~2 mo of age (Fig. 9). Our experiments were conducted on ~50-day-old mice (after the onset of muscle necrosis); therefore, we cannot rule out the likely possibility that merosin is a load-bearing element in skeletal muscles. There are two possible pathways for force transmission that include merosin at the sarcolemmal membrane and through which forces are transmitted between the cytoskeleton and the extracellular space (Fig. 10). Thus a deficiency of merosin should alter the transmission of force in either the axial or transverse plane of the muscle fibers. Lack of merosin disrupts both the transverse and longitudinal pathways of force transmission shown in Fig. 10, and such disruption in force transmission could be responsible at least in part for the severe phenotype and early death in the dy/dy mice. Disruption of these pathways in the dy/dy mouse is consistent with our results on the contractile properties of the merosin-deficient diaphragm that indicate that, regardless of the presence or absence of transverse passive muscle force, maximal muscle tetanic stress is depressed in these mice compared with controls.

View larger version (51K): [in this window] [in a new window]   Fig. 10.   Potential force transmission pathways in skeletal muscles. This schematic presents 2 pathways for force transmission. Pathway 1 includes the structural elements that are situated along the muscle fibers. These structural elements are collagen, merosin m-chain (M), dystroglycans B-subunit (DB), dystroglycans A-subunit (DA), sarcoglycan complex (, , , , ), dystrophin (DY), actin, and z-disks. Pathway 2 consists of structural elements situated in the transverse plane to the muscle fibers. Structural elements in this pathway include collagen, merosin m-chain, integrin A-subunit (IA), integrin B-subunit (IB), talin (T), vinculin (V), desmin (DE), and z-disks. Merosin is clearly visualized in pathway 1, along muscle fibers, and in pathway 2, transverse to muscle fibers. This schematic suggests that loss of merosin may disrupt force transmission pathways in directions along and transverse to the muscle fibers.

Although the depression in muscle force production from dy/dy mice muscles may be attributable to merosin deficiency, the increase in collagen content must also be considered. Collagen fibers and connective tissue do not respond to electric stimulation. That is, these fibers do not display contractile properties. Thus, when merosin-deficient muscles are stimulated, they would be expected to generate a smaller force because of both deficiency of merosin and an increase in collagen content. This is consistent with our model that proposes that merosin is a key component in transmitting the forces both longitudinally and transversely to the muscle fibers.

In summary, our data demonstrate that the passive compliance of either hindlimb and diaphragm muscle is decreased in the absence of merosin. Furthermore, disruption of merosin appears to contribute to the viscous capacity of the diaphragm in response to mechanical loading, specifically, in the transverse plane of the muscle fibers of the diaphragm. Additionally, muscle force is depressed in the merosin-deficient muscles. Taken together, our results suggest that loss of merosin leads to altered muscle passive and active mechanical properties in both longitudinal and transverse pathways of force transmission.