Force transmission, compliance, and viscoelasticity are altered in the {alpha}7-integrin-null mouse diaphragm

M. A. Lopez,1 U. Mayer,2 W. Hwang,1 T. Taylor,1 M. A. Hashmi,1 S. R. Jannapureddy,1 and Aladin M. Boriek1

1Department of Medicine, Baylor College of Medicine, Houston, Texas; and 2Wellcome Trust Center for Cell-Matrix Research, Manchester, United Kingdom

Submitted 26 August 2003 ; accepted in final form 20 August 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
{alpha}7{beta}1 integrin is a transmembrane structural and receptor protein of skeletal muscles, and the absence of {alpha}7-integrin causes muscular dystrophy. We hypothesized that the absence of {alpha}7-integrin alters compliance and viscoelasticity and disrupts the mechanical coupling between passive transverse and axial contractile elements in the diaphragm. In vivo the diaphragm is loaded with pressure, and therefore axial and transverse length-tension relationships are important in assessing its function. We determined diaphragm passive length-tension relationships and the viscoelastic properties of its muscle in 1-month-old {alpha}7-integrin-null mice and age-matched controls. Furthermore, we measured the isometric contractile properties of the diaphragm from mutant and normal mice in the absence and presence of passive force applied in the transverse direction to fibers in 1-month-old and 5-month-old mutant mice. We found that compared with controls, the diaphragm direction of {alpha}7-integrin-null mutants showed 1) a significant decrease in muscle extensibility in 1-year-old mice, whereas muscle extensibility increased in the 1-month-old mice; 2) altered muscle viscoelasticity in the transverse direction of the muscle fibers of 1-month-old mice; 3) a significant increase in force-generating capacity in the diaphragms of 1-month-old mice, whereas in 5-month-old mice muscle contractility was depressed; and 4) significant reductions in mechanical coupling between longitudinal and transverse properties of the muscle fibers of 1-month-old mice. These findings suggest that {alpha}7-integrin serves an important mechanical function in the diaphragm by contributing to passive compliance, viscoelasticity, and modulation of its muscle contractile properties.

muscular dystrophy; respiratory muscles; transmembrane proteins


{alpha}7{beta}1 INTEGRIN IS A TRANSMEMBRANE structural protein that is highly concentrated in myotendinous junctions (MTJ) and costameres of skeletal muscles (2, 19, 31, 38). {alpha}7-Integrin is also found along the entire sarcolemma and at myomyonal junctions in series-fibered muscles (34). Therefore, {alpha}7-integrin could be involved in force transmission not only between the ends of muscle cells and the tendon but also between the axial and lateral elements of the muscle. {alpha}7-Integrin is a receptor for the laminin family of the basement membrane proteins found in the extracellular matrix (8, 21, 35, 41). Absence of the {alpha}7-integrin subunit leads to degenerative disorders of skeletal muscles and disrupts a potential force transmission pathway through the integrin complex (17, 19). In addition, a novel form of congenital muscular dystrophy with normal expression of merosin and deficient levels of {alpha}7-integrin has been documented in humans (19). A previous study (11) showed that the absence of {alpha}7-integrin in mice does not alter expression of the associated dystroglycan proteins and the extracellular protein merosin, both of which provide links to the sarcolemmal membrane (23).

The diaphragm muscle functions in a three-dimensional mechanical environment in which muscle contractile force can be transmitted across the lateral surfaces of the muscle cell, in the transverse plane of the cell, and at the MTJ at the ends of the muscle cell parallel to the axial direction of the cell (4). Most materials of two-dimensional sheets require a larger tensile force to hold a sample at a given stretched length when there is an additional tensile force acting perpendicular to it, in the plane of the sheet, than under uniaxial conditions, where the perpendicular direction is unrestrained (15). The diaphragm is mechanically loaded with pressure in vivo and therefore is subjected to a biaxial stress. That is, the muscle sheet of the diaphragm is subjected to stresses not only in the longitudinal direction but also in the transverse direction of the muscle fibers. Therefore, measuring the length-tension relationship in response to mechanical forces only in the direction of the diaphragm muscle fibers is not sufficient to fully characterize the mechanical properties of the diaphragm. The effect of transverse passive mechanical stress on contractile force production in the diaphragm muscles of normal mice and in those of mice lacking the key transmembrane protein integrin could be important to the understanding of the mechanisms of force transmission. We therefore wondered whether the {alpha}7-integrin plays any role in mediating the transmission of contractile force, possibly through the transverse direction of the muscle fibers of the diaphragm.

We hypothesized that the absence of {alpha}7-integrin might alter diaphragm muscle compliance and viscoelasticity as well as disrupt contractile force transmission between the longitudinal and transverse axes of the diaphragm muscle. To test this hypothesis, we used diaphragm muscles from {alpha}7-integrin-null mice and measured their passive length-tension relationships. Furthermore, we measured the stress-relaxation curves and muscle contractile properties of the diaphragm in the presence and absence of passive transverse mechanical stress. We found that the diaphragm muscles of the {alpha}7-integrin-null mutants showed 1) a significant increase in diaphragm muscle extensibility in 1-month-old mice, whereas muscle extensibility was decreased in 1-year-old mice; 2) altered muscle viscoelasticity in the transverse plane of the diaphragm in 1-month-old mice; 3) a significant increase in the force-generating capacity of the diaphragm in 1-month-old mice, whereas muscle contractility was depressed in 5-month-old mice; and 4) reduction in mechanical coupling between longitudinal and transverse properties of the diaphragm muscle sheet in 1-month-old mice.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Mice and tissue preparation. The experimental protocols for this study used 26 129/SvJ {alpha}7-integrin-null mice and 24 normal wild-type 129/SvJ mice weighing 16–24 g. These mice were maintained in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and procedures were approved in advance by the Institutional Review Board of Baylor College of Medicine. The mice were anesthetized with an intravenous injection of pentobarbital sodium. The diaphragm muscle was excised and immediately immersed in a muscle bath containing a modified Krebs-Ringer solution (in mM: 137 NaCl, 5 KCl, 1 NaH2PO4, 24 NaHCO3, 2 CaCl2, and 1 MgSO4, pH 7.4) bubbled with 95% O2-5% CO2 (9). The solution was maintained at 25°C throughout the experiments. Two pairs of markers made of silk surgical thread (0.2 mm) were sutured to the surface of the muscle in a centrally located position away from the insertions in a square pattern at 1-mm intervals on the membranes of two neighboring muscle fibers. The markers were consistently sutured onto the abdominal surface of the midcostal region of the left hemidiaphragm.

Muscle mechanical testing. Muscle was placed into an in vitro biaxial muscle mechanical testing apparatus. This apparatus had two orthogonal axes, and each axis was driven by a micrometer. Four small, identical alligator clamps, one for each side of the muscle, were used to hold the muscle during passive lengthening and passive shortening maneuvers. The diaphragm muscle was secured by fixing one clamp to the central tendon and the opposing clamp to the tendon at the chest wall insertion, with the lower three ribs intact. The muscle was mounted so that the position of the markers on the surface of the muscle sheet during stretching and shortening could be viewed using a black-and-white closed-circuit television-type camera and recorded on a videocassette tape. The recorded videotape was digitally captured with a video capture card using a frame grabber at a sampling rate of 2 Hz. The markers on the surface of the muscle were then digitized using Image Tool version 2.0 software (http://ddsdx.uthscsa.edu/dig/itdesc.html). The precise marker position on a Cartesian coordinate axis was determined, assuming that all markers were within the same plane. Two force transducers (model FORT 100, ±50 g, differential bridge type; World Precision Instruments, Sarasota, FL) were positioned on orthogonal axes. This set of force transducers was used to measure passive muscle force, and each transducer had a resolution of 0.01% full scale, a sensitivity of 3 µV/V/g, and a hysteresis of 0.1% full scale. The measured forces were then amplified and recorded using LabVIEW software (National Instruments, Austin, TX).

Measurements of length-tension relationship of the diaphragm. The in vitro passive mechanics experiments were conducted using 10 normal 129/SvJ mice (weight, 20.35 ± 1.91 g; age, 37 ± 5 days) and 8 {alpha}7-integrin-null 129/SvJ mice (weight, 16.51 ± 1.53 g; age, 38 ± 5 days). To investigate the effect of age on the length-tension relationship of the diaphragm muscle, we used four normal 129/SvJ mice (weight, 31.5 ± 1.8 g; age, 345.3 ± 4.6 days) and four additional {alpha}7-integrin-null 129/SvJ mice (weight, 23.1 ± 2.6 g; age, 372 ± 18 days). It is important to note that at age 30 days, the homozygous mouse was healthy and appeared essentially indistinguishable from the age-matched normal mouse. By about 1 yr of age, despite normal feeding habits, the homozygous mutant mouse had lost about half of its weight and showed abnormal posture characterized by hunching (Fig. 1).



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Fig. 1. A: at 30 days of age, the homozygous {alpha}7–/– mouse was healthy and appeared indistinguishable from the age-matched wild-type control. B: by 1 yr of age, despite normal feeding habits, the homozygous {alpha}7–/– mouse had lost ~50% of its weight and had abnormal posture characterized by hunching.

 
Optimal length (Lo) was obtained by determining the length at which the muscle produced maximal twitch force in response to electrical stimulation. Lo was determined to correspond to a tension of ~5 g/cm for both normal and dystrophic muscles. To establish a constant history of mechanical loading, the muscle was preconditioned with five cycles of passive lengthening to Lo and then passive shortening to the unstressed length. Length-tension curves were then measured by passively lengthening the muscle in the longitudinal direction of the muscle fibers from unstressed length to a longer length that corresponded to an applied tension of ~15 g/cm, followed by passive shortening of the muscle to the unstressed length. Length-tension curves were also measured in the direction transverse to the muscle fibers by lengthening the muscle in the direction transverse to the muscle fibers, followed by passive shortening to the unstressed state. Lengthening the muscle in the transverse direction was performed by applying either 1 or 2 g of force in the plane of the muscle sheet orthogonal to the muscle fibers. These are equivalent to one-sixth or one-third maximal tetanic stresses that could be generated by the muscle under isometric conditions.

Computation of two-dimensional muscle strains. Planar mechanical strains on the surface of the muscle sheet were calculated according to modified methods used in a previous in vivo mechanical strain analysis of the canine diaphragm at our laboratory (7). Briefly, the region enclosed by the markers at the center of the diaphragm muscle sheet was divided into triangles, with each set of three adjacent markers defining the apices of the triangles. Any three markers defining a triangle constitute a plane. The Cartesian coordinates of these markers in the plane at the reference position were denoted xi and yi (i = 1, 2, 3). The displacements of the markers from their reference positions to their positions at a deformed state were denoted ui and vi and were assumed to be a linear function of position. The following equation:

(1)

with the measured displacement (ui) and position coordinates (xi and yi) provided a set of three equations for three coefficients: a1, a2, and a3. Similarly, the data provided the information required to determine the values of the coefficients (a4, a5, and a6) in the following equation:

(2)

Shear strains were small, and therefore the values of {epsilon}x and {epsilon}y essentially reflected strains in the directions parallel to the muscle fibers and transverse to the fibers, respectively. Mechanical strains {epsilon}x and {epsilon}y, which occurred during either passive lengthening or passive shortening of the muscle sheets, were calculated according to the small-strain elasticity theory (16):

(3)

(4)

The values {delta}u and {delta}v denote the displacement of the markers on the surface of the diaphragm in the x (parallel to muscle fibers) and y (transverse to muscle fibers) directions, respectively. The coefficients a2, a3, a5, and a6 were substituted in Equations 3 and 4 to obtain {delta}u/{delta}x, {delta}v/{delta}y. Mechanical strains were computed relative to the unstressed length of the muscle. Unstressed length was defined as the length of the muscle sheet placed into the muscle bath and attached to the clamps in the absence of applied mechanical forces. The muscle extensibility ratio ({lambda}) was computed as follows: {lambda} = 1 + {epsilon}, where {epsilon} is the strain either in the muscle fiber or transverse to the fiber direction.

Measurement and modeling of viscoelastic properties. We used eight {alpha}7-integrin-null 129/SvJ mice (weight, 20.4 ± 4.48 g; age, 47.0 ± 9.74 days) and seven normal wild-type 129/SvJ mice (weight, 21.3 ± 2.78 g; age, 40.4 ± 6.21 days). After producing the length-tension curves in either the longitudinal or the transverse direction of the muscle fibers, we measured stress-relaxation curves. The viscoelastic behavior of the living tissue was sensitive to the history of mechanical deformation. Therefore, muscles were initially preconditioned with five cycles of passive lengthening of the tissue to optimal length followed by mechanical unloading of the muscle to the unstressed length. The muscle was then stretched to optimal length and maintained at that length. Muscle force was allowed to relax asymptotically until it essentially reached a plateau. Forces were measured at 10 Hz using a force transducer (model FORT 100, ±50 g; World Precision Instruments).

We used nonlinear least-squares routines created in MATLAB software to fit the stress-relaxation data to the classical standard linear solid model of viscoelasticity (16). This simple model describes the muscle as a parallel combination of a dashpot with coefficient of viscosity {eta}1 and a linear spring with spring constant µ1, with a second linear spring with spring constant µ0 parallel to the first spring and the dashpot. The dashpot consists of a cylinder holding a piston immersed in viscous fluid, and the fit of the piston and cylinder is not tight. The piston moves slowly in response to mechanical applied load, and the higher the loading, the faster the piston moves. The dashpot relaxation time is the length of time necessary for the dashpot, or the shock-absorbing quality of the muscle, to essentially be relaxed, and at this point, the muscle is characterized as perfectly elastic. The relaxation function based on this model is of the following form:

where F is the relaxation force, t is time, ER is the relaxed elastic modulus, {tau}{epsilon} is relaxation time for constant strain, and {tau}{sigma} is the relaxation time for constant stress (16). After obtaining the three constants ER, {tau}{epsilon}, and {tau}{sigma}, we calculated the viscoelastic coefficients {eta}1, µ1, and µo on the basis of the force-displacement relationships of the viscoelastic model. The {eta}11 ratio is a relaxation time. We report the relaxed elastic modulus and the dashpot relaxation time ({eta}11).

Measurement of isometric contractile properties of the diaphragm. For these experiments, we used two age groups of mice. The first group consisted of 7 normal 129/SvJ mice (weight, 17.4 g; age, 30 ± 1.1 days) and 10 {alpha}7-integrin-null 129/SvJ mice (weight, 14.8 g; age, 30 ± 0.8 days). The second group consisted of four normal 129/SvJ mice (weight, 30.1 ± 2.7 g; age, 151.5 ± 0.6 days) and five {alpha}7-integrin-null 129/SvJ mice (weight, 20.8 ± 2.9 g; age, 156.7 ± 0.6 days). After anesthetizing the mice, we excised the left hemidiaphragms and immediately placed them into a muscle bath with continuously circulating oxygenated 95% O2-5% CO2 Krebs-Ringer solution maintained at 25°C. Muscles were clamped on both the central tendon and the tendon at the chest wall insertion, with one clamp connected to a Cooper Instruments LQB 630 force transducer (Cooper Instruments, Warrenton, VA) and the other clamp attached to a force carriage. After positioning the muscle between two stainless steel mesh electrodes, we electrically stimulated the muscle using a Grass S88 stimulator (Grass-Telefactor, West Warwick, RI). Muscles were stimulated directly using supramaximal current intensity (measured characteristics: 550 mA at 12 V). Optimal length was determined by twitch responses (0.1-ms stimulus duration). We used stimulus durations that evoked maximal twitch response (0.5 ms at 25°C) and tetanic train durations that produced an unambiguous plateau of tetanic force (0.5-ms pulses, tetanic train duration of 500 ms, 120-s recovery). The muscle was then clamped in the transverse direction to the long axis of the muscle fibers, with one clamp attached to a World Precision Instruments FORT 250 force transducer and the other clamp connected to a force carriage. Tetanic stimulation sequences were repeated three times in the absence of any transverse passive forces and in the presence of either 1- or 2-g passive forces applied in the direction transverse to muscle fibers. Contractile force data were collected at a sample rate of 300 Hz using 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.

Muscle contractile stress was computed as the ratio of active muscle tension to the unstressed thickness (stress = tension/thickness), with stress expressed in Newtons per square centimeter, tension assessed in Newtons per centimeter (computed as measured force in grams divided by muscle clamp width), and muscle thickness measured in centimeters. To measure muscle thickness, a digital image of the surface of the muscle sheet was obtained. The muscle was then gently blotted dry and weighed, and the surface area of the muscle sheet was determined using Image Tool version 2.0. Muscle thickness was computed using the measured surface area, mass, and muscle density. MATLAB version 5.0 software (MathWorks, Natick, MA) was used to analyze maximal tetanic data.

Statistical analysis. Differences between groups were assessed by performing ANOVA with the use of the SAS software program (27). The model used was a two-factor effects model for two groups of mice ({alpha}7-integrin-null vs. normal) and two treatments (passive mechanics: uniaxial stretch parallel to the muscle fibers vs. uniaxial stretch transverse to fibers; muscle contractility: uniaxial loading vs. either form of the biaxial loading). Muscle samples were handled as a random effect; force, treatment, and force-by-treatment interaction were handled as fixed effects. Pairwise comparisons were performed to test a priori hypotheses using linear contrast. Data are expressed as means ± SE, where SE is the standard error unless otherwise indicated. Comparisons were considered significant at P < 0.05.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Passive length-tension relationships of diaphragm muscles from 1-month-old {alpha}7-integrin-null and their age-matched normal wild-type controls are shown in Fig. 2. Data were obtained from a representative {alpha}7-integrin-null 129/SvJ mouse (weight, 13.6 g; age, 34 days) and compared with data from a representative normal 129/SvJ mouse (weight, 21.6 g; age, 33 days). For either diaphragm, passive tension increased continuously in a nonlinear fashion. However, the nonlinear behaviors in the two directions were quite different. The length-tension relationship in the muscle direction was only mildly nonlinear over the sizable range of stretches represented in our data. The nonlinear stiffening in the transverse direction was stronger, and a sharp stop in muscle extensibility occurred at a small transverse tension. Furthermore, hysteresis was exhibited in the direction of the muscle fibers, whereas it was essentially negligible in the transverse direction to the muscle fibers. Compared with control, the diaphragm length-tension relationship of the mutant mouse was shifted to the right, indicating increased muscle extensibility in the {alpha}7-integrin-null diaphragm. Lack of {alpha}7-integrin did not appear to alter muscle anisotropy in the diaphragm. That is, muscle appeared stiffer in the direction transverse to the fibers than parallel to the fibers in the mutant mice. Using 10 normal wild-type mice and 8 mutant mice, we report the average extension ratio at an applied tension of 5 g/cm. Passive lengthening in the fiber direction resulted in extension ratios that were higher in {alpha}7-integrin-null mice ({alpha}7-integrin–/–: {lambda} = 1.34 ± 0.17, {alpha}7-integrin+/+: {lambda} = 1.19 ± 0.04; P < 0.05). Passive lengthening transverse to the fiber resulted in an extension ratio for {alpha}7-integrin-null mice that was essentially the same as that for controls ({alpha}7-integrin–/–: {lambda} = 1.04 ± 0.04 vs. {alpha}7-integrin+/+: {lambda} = 1.04 ± 0.04).



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Fig. 2. Representative length-tension relationships of the diaphragm muscles of 1-month-old {alpha}7-integrin+/+ and age-matched {alpha}7-integrin–/– mice. The diaphragm muscles were passively lengthened either in the longitudinal direction or in the transverse direction to the muscle fibers. The longitudinal length-tension curves for the mutants and controls are indicated by the symbols ({square}) and ({circ}), respectively. The transverse length-tension curves for the mutants and controls are indicated by the symbols (+) and ({bullet}), respectively. Compared with the control muscle, the longitudinal and transverse length-tension curves of the diaphragm muscle from the mutant mouse are shifted to the right. This implies that the diaphragm muscle of the mutant is more extensible than that of the control mouse. The increased muscle extensibility appears to be more pronounced in the longitudinal direction than in the transverse direction to the muscle fibers.

 
Passive length-tension relationships in the diaphragm muscles from a representative 1-year-old {alpha}7-integrin-null mouse (weight, 24.3 g; age, 330 days) and a representative age-matched wild-type normal mouse (weight, 32 g; age, 346 days) are shown in Fig. 3. Length-tension relationships of the 1-year-old {alpha}7-integrin-null mouse are shifted to the left compared with those of the wild-type normal mouse. At a tension of 5 g/cm, the extension ratios in the direction of the muscle fibers were as follows: 1-year-old {alpha}7-integrin-null: {lambda} = 1.21 ± 0.05 vs. 1-year-old normal wild type: {lambda} = 1.36 ± 0.03. In the direction transverse to the muscle fibers, the corresponding values were as follows: {alpha}7-integrin-null: {lambda} = 1.04 ± 0.01 vs. normal wild type: {lambda} = 1.02 ± 0.01. These data suggest that passive muscle compliance of the 1-year-old {alpha}7-integrin-null normal diaphragm was significantly decreased compared with that of the diaphragms from age-matched normal wild-type mice.



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Fig. 3. Representative length-tension relationships along the muscle fibers and transverse to the muscle fibers for diaphragm muscles from a 1-year-old 129SV {alpha}7-integrin-null mouse and a 1-year-old wild-type 129SV {alpha}7-integrin+/+ mouse. The diaphragm muscles were lengthened and shortened passively in the directions along and transverse to the fibers. The symbols ({circ}) and ({bullet}) indicate the length-tension relationships along the muscle fibers and transverse to the fibers for the wild-type mouse diaphragm. The symbols ({square}) and (+) show the length-tension curves in the {alpha}7-integrin-null diaphragm in the directions along the muscle fibers and transverse to the fibers, respectively. The length-tension relationships of the 1-year-old {alpha}7-integrin-null mouse diaphragm are shifted to the left of that of the 1-year-old wild-type mouse diaphragm. At a tension of 5 g/cm, the extension ratio in the direction of the muscle fibers for the {alpha}7-integrin-null mouse and 1-year-old wild-type mouse diaphragms are 1.21 and 1.36, respectively. At the same level of tension, the extension ratios in the transverse direction to the muscle fibers for the {alpha}7 integrin-null mouse and the wild-type mouse are 1.04 and 1.02, respectively. These data suggest that compared with control, passive muscle compliance of the diaphragm is decreased in the old mutant mice. The decreased compliance appears to be more pronounced in the longitudinal direction of the muscle fibers.

 
Representative length-tension relationships of the diaphragm muscle from a 1-month-old mouse compared with the diaphragm muscle of a 1-year-old mutant mouse are shown in Fig. 4. Each muscle was passively lengthened and shortened in the direction of the muscle fibers. Muscle length was computed as a percentage of the unstressed length. The length-tension curve of the 1-month-old mutant mouse was shifted to the right compared with that of the 1-year-old mutant mouse. Having studied eight mutant mice at age 1 month and five mutant mice at age 1 year, we report the average extension ratio at passive tension of 5 g/cm. The extension ratios of the diaphragms from mutant mice at ages 1 month and 1 year were {alpha}7-integrin–/– 1 mo old: {lambda} = 1.15 ± 0.02 vs. {alpha}7-integrin–/– 1 yr old: {lambda} = 1.07 ± 0.015 (P < 0.05). Passive lengthening transverse to the fiber resulted in similar extension ratios for {alpha}7-integrin–/– murine diaphragm and the normal murine diaphragm ({alpha}7-integrin–/–: {lambda} = 1.02 ± 0.02 vs. {alpha}7-integrin+/+: 1.04 ± 0.02; P < 0.25). These data demonstrate that muscle extensibility and compliance in the direction of the muscle fibers of the {alpha}7-integrin-null murine diaphragm decreased with age.



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Fig. 4. Representative length-tension relationships of the diaphragm muscles of representative 1-month-old and 1-year-old {alpha}7-integrin–/– mutant mice. The diaphragm muscles were passively lengthened and shortened in the directions along and transverse to muscle fibers. The length-tension curves in the longitudinal direction of the muscle fibers of the 1-year-old and the 1-month-old mutant mice are depicted by symbols ({circ}) and ({square}), respectively. The transverse length-tension curves of the 1-year-old and 1-month-old mice are depicted by the symbols ({bullet}) and (+), respectively. The length-tension relationship in the muscle fiber direction of the 1-month-old mouse is shifted to the right compared with that of the 1-year-old mouse. The length-tension curves in the transverse direction to the muscle fibers appear similar. These data demonstrate that the diaphragm of the 1-year-old {alpha}7-integrin-null mouse is less extensible in the direction of the muscle fibers than that of the 1-month-old {alpha}7-integrin-null mouse. Therefore, the mechanical properties of the passive diaphragm muscle in the direction of the muscle fibers are significantly altered by age in the {alpha}7-integrin-null mouse.

 
Using the standard linear solid model, we determined the relaxed elastic modulus for {alpha}7-integrin-null and normal mouse diaphragm muscles. ER provides a quantitative value of the relaxed stiffness of the muscle. An ER value of 1.0 would indicate a 100% elastic material, i.e., a material characterized only by linear springs and having no dashpot component. The data in Fig. 5A show ER values for young {alpha}7-integrin-null diaphragms compared with those of age-matched controls. For controls, ER was greater in the direction transverse to the muscle fibers (P < 0.04). The difference in ER between {alpha}7-integrin-null diaphragms and control diaphragms was significant only in the direction transverse to the muscle fibers (P < 0.04). The data suggest that relaxed stiffness is altered by a deficiency in {alpha}7-integrin. Fitted stress-relaxation curves with corresponding raw data are shown for {alpha}7-integrin-null and normal diaphragms in Fig. 5B. The two curves represent passive force data collected when each muscle was kept at a constant stretched state in the direction transverse to the muscle fibers for {alpha}7-integrin-null and normal diaphragm. It is important to note that these curves are not collective averages but data from individual diaphragms representative of the mean response. In the direction transverse to the diaphragm muscle fibers, the ER values were greater in normal murine diaphragm muscle than in {alpha}7-integrin-null diaphragm muscle (P < 0.005). Dashpot relaxation time was not significantly different between {alpha}7-integrin-null and control diaphragms (data not shown).



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Fig. 5. A: means ± SE of relaxed elastic modulus (ER) values for {alpha}7-integrin-null diaphragms compared with controls are shown. Data were obtained from eight {alpha}7-integrin-null mutant mice (weight: 20.4 ± 4.48 g; age: 47.0 ± 9.74 days) and seven normal mice (weight: 21.3 ± 2.78 g; age: 40.4 ± 6.21 days). For controls, ER values along and transverse to the fibers were 0.59 ± 0.05 and 0.68 ± 0.02, respectively, with a higher ER value in the transverse plane (*P < 0.04). For the diaphragm muscles of {alpha}7-integrin-null mice, ER values parallel and transverse to the fibers were 0.61 ± 0.05 and 0.57 ± 0.04, respectively. The difference in ER between {alpha}7-integrin-null mice and controls was greater in the direction transverse to the muscle fibers ({varphi}P < 0.04). B: fitted stress-relaxation curves with corresponding raw data are shown for representative {alpha}7-integrin-null and normal diaphragms. The two curves represent the loading transverse to the muscle fibers for {alpha}7-integrin-null and normal mice. Raw ({circ}) and fitted data (–) are shown. These curves are not collective averages but data for individual mice that are representative of the mean. ER values are an indication of muscle stiffness; a small ER value reflects large muscle compliance. The ER values were greater for normal mouse diaphragms than for {alpha}7-integrin-null diaphragms in the direction transverse to the muscle fibers. P < 0.005. Data in A and B suggest altered viscoelastic behavior of the diaphragm muscle from the {alpha}7-integrin-null mouse compared with that of the wild-type control mouse in the transverse plane.

 
Data regarding maximum tetanic stress of diaphragms from 1-month-old {alpha}7-integrin-null mice and age-matched wild-type control mice are shown in Fig. 6. Data were obtained from 7 normal 129/SvJ mice (weight, 17.4 g; age, 30 ± 1.1 days) and 10 {alpha}7-integrin-null 129/SvJ mice (weight, 14.8 g; age, 30 ± 0.8 days). Surprisingly, maximal muscle tetanic stress was enhanced in the {alpha}7-integrin-null diaphragm compared with the normal diaphragm only under uniaxial loading. Biaxial loading increased muscle stress only in the normal diaphragm. To determine whether age had an effect on force production and force transmission of the diaphragm muscle, we measured maximal tetanic stress in normal and {alpha}7-integrin-null diaphragms of 5-month-old mutant mice and their age-matched normal wild-type controls. Contractile force was measured under uniaxial and biaxial states of mechanical loading. The data shown in Fig. 7 were obtained from four normal 129/SvJ wild-type mice (weight, 30.1 ± 2.7 g; age, 151.5 ± 0.6 days) and five {alpha}7-integrin-null 129/SvJ mice (weight, 20.8 ± 2.9 g; age, 156.7 ± 0.6 days). Compared with controls, maximal tetanic muscle stress under all loading conditions was significantly less in the {alpha}7-integrin-null murine diaphragms. In these older mice, transverse passive mechanical stress appeared to increase muscle contractile force in both normal and {alpha}7-integrin-null diaphragms. These data demonstrate that diaphragm muscle contractility was depressed in the older {alpha}7-integrin-null mice.



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Fig. 6. Maximum tetanic stress in diaphragm muscle from {alpha}7-integrin+/+ and {alpha}7-integrin–/– mice. Data were obtained from 7 normal 129/SvJ mice (weight, 17.4 g; age, 30 ± 1.1 days) and 10 {alpha}7-integrin-null 129/SvJ mice (weight, 14.8 g; age, 30 ± 0.8 days). Muscle stress was enhanced in the {alpha}7-integrin-null mouse diaphragm compared with that in the normal mouse only under uniaxial loading condition. Biaxial loading increased muscle stress only in the normal mouse diaphragm. That is, force transmission was compromised in the {alpha}7-integrin-null mouse diaphragms. These data suggest an important mechanical role of {alpha}7-integrin; it participates in the transmission of contractile muscle force between adjacent myofibrils.

 


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Fig. 7. Maximum tetanic stress in diaphragm muscle from {alpha}7-integrin+/+ and {alpha}7-integrin–/– mice. Data are from 4 normal 129/SvJ mice (weight, 30.1 ± 2.7 g; age, 151.5 ± 0.6 days) and 5 {alpha}7-integrin-null 129/SvJ mice (weight, 20.8 ± 2.9 g; age, 156.7 ± 0.6 days). Maximum tetanic stress was depressed in the {alpha}7-integrin-null diaphragm compared with that in the diaphragms from normal mice under all loading conditions. Biaxial load appeared to enhance muscle stress in the normal and {alpha}7-integrin-null mouse diaphragms.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this study, we have demonstrated the following in {alpha}7-integrin-null mice compared with age-matched wild-type controls: 1) there was a significant decrease in passive muscle extensibility in the 1-year-old mutants, whereas muscle extensibility increased in the 1-month-old mutants; 2) the relaxed elastic modulus was decreased in 1-month-old mice in the direction transverse to muscle fibers; 3) the force-generating capacity in the diaphragms increased significantly in 1-month-old mutants but decreased significantly in 5-month-old mutants; and 4) the mechanical coupling between longitudinal and transverse properties in the diaphragms of the mutant mice was disrupted at age 1 mo.

Integrins are important structural and receptor proteins because they provide one of the major ways in which cells can both bind and respond to the extracellular matrix (1, 8, 29). Integrins participate in vital biological processes such as embryonic development, cell differentiation, maintenance of tissue integrity, adhesion function, and cell-extracellular matrix interaction (10, 17, 22, 24, 26). {beta}1-Integrin is expressed throughout the body, while {alpha}7-integrin is tissue-specific for both skeletal and cardiac muscles. Different isoforms of {alpha}7-integrin appear to have distinct functions in skeletal muscles. Alternative forms of the {alpha}7-integrin (13, 36, 42) are expressed in a developmentally regulated fashion during myogenesis, and different {alpha}7{beta}1-isoforms localize at specific sites on myofibers (28). {alpha}7-integrin was found highly concentrated in MTJ (2, 31, 38). It is known that the MTJ is the principal structure that transmits force generated by muscle contraction to the tendon (39, 40). Furthermore, Mayer et al. (30, 32) have demonstrated that histological analysis of skeletal muscles from mice lacking {alpha}7-integrin revealed typical signs of a progressive muscular dystrophic phenotype in the vicinity of their MTJ. Therefore, their findings are consistent with {alpha}7-integrin being an important linkage between the muscle fiber and the extracellular matrix (25). Furthermore, because {alpha}7-integrin was also found along the entire sarcolemma (34), it is possible that {alpha}7-integrin could contribute to longitudinal and lateral force transmission between myofibers and the extracellular matrix. Our functional data demonstrating lack of the mechanical effect of transverse passive stress on muscle contractility of the diaphragm in the young mutant mice is consistent with this possibility. Mayer et al. (30) reported that the first histopathological changes in the diaphragm of {alpha}7-integrin-null mice were detected at 60 days of age with a few foci of fiber degeneration and were obvious after 100 days. Despite strong histological abnormalities of the diaphragm, {alpha}7-integrin-null mice show no overt respiratory problems before 100 days of age, as was also reported for mdx mice (37).

Our data demonstrate that muscle compliance in the diaphragms of young mutant mice appears to increase compared with the diaphragms of normal wild-type mice. These data are consistent with the possibility of {alpha}7-integrin's being a load-bearing protein. Our data also show that diaphragm muscle compliance is significantly decreased in 1-year-old {alpha}7-integrin-null mice compared with age-matched controls. Because the onset of muscle necrosis in the {alpha}7-integrin-null diaphragm occurs at ~100 days of age, the decrease in muscle compliance in these diaphragms could possibly be due to muscle necrosis and the possible upregulation of collagen. Increased collagen and fibrosis are general features of muscular dystrophy, and decreased muscle compliance has generally been linked to increased collagen content (3, 14, 18, 33, 37). Some studies, however, have shown that increased collagen is not necessarily correlated with muscle compliance. In particular, alteration in muscle compliance was not associated with increased collagen due to aging in the rat soleus muscle. Furthermore, Coirault et al. (12) demonstrated increased muscle compliance of the Syrian hamster diaphragm, while the surface area of collagen was increased. Therefore, the reduced muscle compliance that we have observed in older {alpha}7-integrin-null mouse diaphragms is not necessarily due to upregulation of collagen.

In an earlier study, we (4) investigated the mechanical role of desmin, a cytoskeletal protein, in muscles. In that study, it was demonstrated that desmin integrated the three-dimensional properties of skeletal muscles by coupling the longitudinal and transverse mechanical properties of the diaphragm. The present study and the previous study conducted by Boriek et al. (4) have demonstrated that transverse loads increase maximal muscle contractile force production in the normal diaphragm, the presence of structures that couple the diaphragm muscle's longitudinal and transverse properties of the diaphragm muscle. Our mechanical data obtained in 1-month-old mice suggest that desmin may not be the only structural protein that integrates the longitudinal and transverse elements in skeletal muscles. The integrin complex may be another structural element that could transmit muscle force between the longitudinal and transverse elements. This hypothesis is consistent with recent data demonstrating that {alpha}7-integrin is expressed along the entire sarcolemma and at myomyonal junctions in series-fibered muscles (34).

With regard to viscoelastic behavior, the force needed to maintain a particular constant extension once that extension has been produced (or the stress needed to maintain a particular constant strain) decreases over time. Using the standard linear viscoelastic model, we started by only extending the series spring. As time passed, that spring relaxed while the parallel spring extended to share the load. The reduction of stress at constant strain is known as stress relaxation, and the ratio of stress to strain is known as the relaxation modulus, which decreases over time. Stress relaxation is a characteristic of an elastic sheet that displays a viscous response. Previous studies of the diaphragm have shown that this muscle displays a viscous response (4). Measurements of the viscoelastic properties include ER values, which provide a quantitative value for the degree of relaxed stiffness in muscle; an ER of 1.0 signifies a perfectly elastic material. The results of the present study show that ER values are greater in the direction transverse to the fibers than those in the longitudinal direction. This finding is consistent with the diaphragm muscle being stiffer in the direction transverse to the muscle fibers than along the muscle fibers. These data are consistent with passive mechanical data found in the diaphragms of the dog (5) and the rat (6).

In 1-month-old mice, maximal muscle tetanic stress was enhanced in the {alpha}7-integrin-null diaphragm compared with the normal diaphragm only under the uniaxial loading condition. Biaxial loading increased muscle stress only in the normal diaphragm. The increase in muscle force in the {alpha}7-integrin-null mice is consistent with a possible role for {alpha}7-integrin in dissipating mechanical energy of the diaphragm during normal breathing. However, in young mice, the increase in tetanic stresses that resulted from biaxial mechanical loading of diaphragm appeared to require, at least in part, the contribution of {alpha}7-integrin.

Cohn et al. (11) provided a quantitative assessment of the sarcolemmal immunoreactivity of muscle proteins in {alpha}7-integrin mutant mice. They found that other transmembrane proteins of the dystroglycan complex and the sarcoglycan complex were unaffected by the absence of {alpha}7-integrin. In addition, normal levels of {alpha}2-laminin appear to be expressed in the mutant mouse. Therefore, our experiments—at least those in young mice—provide data directly pertinent to the mechanical role of {alpha}7-integrin in skeletal muscle function.

{alpha}7{beta}1-Integrin is related to several neuromuscular diseases. Hodges et al. (20) suggested that the expression of {alpha}7-integrin is upregulated in skeletal muscles from either patients with Duchenne muscular dystrophy or an animal model, the mdx mouse. Downregulation of expression of the integrin contributes to muscle pathology in congenital laminin deficiencies. Furthermore, mutations in the {alpha}7-integrin gene have been found to be responsible for a form of congenital neuromuscular disease (19). Burkin and Kaufman (8) overexpressed the {alpha}7-integrin chain in mice that were null in both dystrophin and utrophin, and they found that the transgenic expression of the {alpha}7-integrin chain in these mice ameliorated the development of disease and maintained longevity in mice that otherwise would have developed severe myopathy and died. These investigators suggested that bolstering the integrin-mediated association of muscle with the extracellular matrix could possibly prevent Duchenne muscular dystrophy.

In summary, our data demonstrate that {alpha}7-integrin may contribute to muscle compliance. In addition, the absence of {alpha}7-integrin in 1-month-old mice increased maximal tetanic force production, whereas in 5-month-old mice, force production was compromised in the mutant mice. Our data confirm that passive transverse force alters the production of maximal contractile properties in normal diaphragm and provide new evidence that this effect is absent in {alpha}7-integrin-null diaphragm. Our mechanical data are consistent with a mechanism of force transmission in the diaphragm that is altered by the loss of the {alpha}7-integrin.


    FOOTNOTES
 

Address for reprint requests and other correspondence: A. M. Boriek, Dept. of Medicine, Baylor College of Medicine, One Baylor Plaza, Suite 520B, Houston, TX 77030 (E-mail: boriek{at}bcm.tmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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