Altered muscle force and stiffness of skeletal muscles in alpha -sarcoglycan-deficient mice

Nisha D. Patel, Suneal R. Jannapureddy, Willy Hwang, Imran Chaudhry, and Aladin M. Boriek

Department of Medicine, Baylor College of Medicine, Houston, Texas 77030


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

alpha -Sarcoglycan (ASG) is a transmembrane protein of the dystrophin-associated complex, and absence of ASG causes limb-girdle muscular dystrophy. We hypothesize that disruption of the sarcoglycan complex may alter muscle extensibility and disrupt the coupling between passive transverse and axial contractile elements in the diaphragm. We determined the length-tension relationships of the diaphragm of young ASG-deficient mice and their controls during uniaxial and biaxial loading. We also determined the isometric contractile properties of the diaphragm muscles from mutant and normal mice in the absence and presence of passive transverse stress. We found that the diaphragm muscles of the null mutants for the protein ASG show 1) significant decrease in muscle extensibility in the directions of the muscle fibers and transverse to fibers, 2) significant reductions in force-generating capacity, and 3) significant reductions in coupling between longitudinal and transverse properties. Thus these findings suggest that the sarcoglycan complex serves a mechanical function in the diaphragm by contributing to muscle passive stiffness and to the modulation of the contractile properties of the muscle.

diaphragm mechanics; force transmission; mechanics of breathing; respiratory muscle mechanics; transmembrane proteins


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

LIMB-GIRDLE MUSCULAR DYSTROPHY (LGMD) is a result of deficiencies in the sarcoglycan complex and is a disorder of skeletal muscles. alpha -Sarcoglycan (ASG) is a transmembrane protein situated along the length of fibers of skeletal and cardiac muscles. ASG is one of at least five glycoproteins that are essential to the function of the sarcoglycan complex, and its deficiency results in the downregulation of other glycoproteins. These sarcoglycans, along with the syntrophins and alpha - and beta -dystroglycans, comprise the dystrophin-glycoprotein complex.

Mutations in the ASG gene cause LGMD type 2D (15), an autosomal recessive disorder. ASG-deficient mice develop progressive muscular dystrophy and, in contrast to other animal models for muscular dystrophy, show ongoing muscle necrosis with age, a characteristic of the human disease. Molecular analysis of the ASG-deficient mice demonstrated that absence of ASG resulted in less pronounced sarcoglycan complex, sarcospan, and a disruption of the alpha -dystroglycan association with membrane recessive disorder (10, 17). ASG is a structural protein that could be a load-bearing element in the plane of the cell membrane. ASG could be similar to the extracellular protein, merosin, in that it can transmit forces, mostly in shear, between the cytoskeleton and the collagen matrix fibers. Therefore, most probably, force transmission pathways in both the longitudinal and transverse direction of the myofibers are disrupted in an ASG-null mouse diaphragm. We wondered whether force production and transmission are altered in skeletal muscles when the sarcoglycan complex is disrupted.

In this study, we hypothesize that disruption of the sarcoglycan complex may alter muscle extensibility and disrupt the coupling between passive transverse elements and axial contractile elements in the diaphragm. To test this hypothesis, we measured passive length tension curves in the directions of the fibers and transverse to the fibers of the muscles of the diaphragm and biceps femoris. Furthermore, we measured the isometric contractile properties of the diaphragm in the absence and presence of passive transverse fiber stress.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and tissue preparation. The experimental protocols for this study utilized forty-three 129/SvJ ASG-null and normal wild-type mice weighing 16-24 g. The mice were anesthetized with an intravenous injection of pentobarbital (0.5-0.7 ml/kg). Either the diaphragmatic muscle or biceps femoris muscle was excised and immediately immersed into a muscle bath containing a modified Krebs-Ringer solution (in mM: 137 NaCl, 5 KCl, 1NaH2PO4, 24 NaHCO3, 2 CaCl2, 1 MgSO4, pH 7.4) bubbled with 95% O2-5% CO2 (7). The solution was maintained at a temperature of 25°C throughout the muscle preparation and experimental phase. We excised either left hemidiaphragms or left biceps femoris. The muscle of the diaphragm included the origin on the central tendon and insertion on the rib cage. The biceps femoris included the entire muscle fibers from origin to insertions on the muscle tendonous junctions. Four silk suture position markers (7-0 or 8-0 Surgilene) were sutured on the surface of the appropriate muscle. All markers were placed in the central region of the muscle to minimize the contribution of the mechanical effects of muscle attachments. The markers were placed in a square configuration ~1 mm apart from each other. Two pairs of marker were aligned in the direction along the fibers and two transverse to the fibers.

Biaxial mechanical muscle testing. The biaxial tissue testing apparatus was used to apply in-plane uniaxial stress to the tissue along the fiber direction, uniaxial stress applied transverse to the fiber direction, or biaxial stress applied in both directions, along and transverse to the muscle fibers. The description of the biaxial testing apparatus is detailed elsewhere (3). Biaxial loading of a muscle sheet refers to the muscle being subjected to mechanical stresses not only in the direction of the muscle fibers but also in the direction transverse to the fibers.

Measurements of muscle passive mechanical properties. Either costal hemidiaphragms or biceps femoris muscles from eight normal wild-type 129/SvJ mice (weight: 23.6 ± 7.2 g; age: 43.5 ± 15.9 days) and seven 129/SvJ ASG-null mice (weight: 17.8 ± 2.6 g; age: 32.3 ± 2.4 days) were used in these experiments. After the mice were anesthetized, the appropriate muscles were quickly submerged in the oxygenated Krebs-Ringer solution. All mechanical loads were applied in the plane of the muscle sheet. We preconditioned each muscle with five uniaxial lengthening-shortening cycles with the peak tension of 5 g/cm, and quasistatic lengthening-shortening cycles characterized the passive length-tension of the muscle. Muscles were then passively lengthened from unstressed length with a peak tension of about 20 g/cm and then passively shortened until passive force was negligible. The uniaxial stretching maneuver consisted of clamping one end of the muscle sheet at a fixed position and stretching the other end of the muscle. Biaxial maneuvers were only applied to the diaphragm muscle and consisted of passive mechanical stretching of the muscle in the direction transverse to the muscle fibers by either 1 or 2 g and then stretching the sheet axially in the muscle in the fiber direction. Stress was computed as applied force divided by cross-sectional area. Therefore, force was divided by the multiplication of muscle width and unstressed muscle thickness [stress = force/(width × thickness)], where stress is in N/cm2.

Computation of two-dimensional strains. The strains in the plane of the muscle were computed by the following procedure. The marked region is divided into triangles with markers forming the apices. The coordinates of these points in that unstressed plane of the diaphragm are denoted xi and yi, (i = 1, 2). The displacement, ui, from the unstressed state to the deformed state is assumed to be a linear function of position and is computed as follows
u<SUB>i</SUB>=a<SUB>1</SUB>+a<SUB>2</SUB>x<SUB>i</SUB>+a<SUB>3</SUB>y<SUB>i</SUB> (1)
This equation with known values of the displacements and the position of three markers substituted for ui, xi, and yi provides a set of three equations for the three coefficients: a1, a2, and a3. Similarly, the data provide the information required for determining the coefficients (a4, a5, and a6) in the following equation
v<SUB>i</SUB>=a<SUB>4</SUB>+a<SUB>5</SUB>x<SUB>i</SUB>+a<SUB>6</SUB>y<SUB>i</SUB> (2)
The values of the coefficients a1, a2, etc., were used to find the partial derivatives which were substituted into the following equations
ϵ<SUB>x</SUB>=&dgr;u/&dgr;x

ϵ<SUB>y</SUB>=&dgr;v/&dgr;y (3)

ϵ<SUB>xy</SUB>=&dgr;u/&dgr;y+&dgr;v/&dgr;x
where delta u and delta v denote the marker's displacement in the x (along fibers) and y (transverse) directions, defined to calculate strains. The strains, varepsilon x and varepsilon y, were computed for each triangle. varepsilon xy is shear strain and is essentially negligible, and, therefore, varepsilon x is in the muscle fiber direction, whereas varepsilon y is in the transverse direction to the fibers.
&lgr;=1+ϵ (4)
where lambda  is the extension ratio and varepsilon  is strain in either the fiber or transverse to fibers direction.

Measurements of contractile properties. Muscles from seven 129/SvJ wild-type mice (weight: 17.36 ± 4.66 g; age: 30 ± 13 days) and eight ASG-null mice (weight: 17.56 ± 1.66 g; age: 30 ± 11 days) were used. Upon anesthetizing of the animals, the diaphragm muscle was excised and placed in the oxygenated muscle bath. The muscle was positioned horizontally between two stainless steel mesh electrodes, and optimal length was determined by twitch responses (0.1-ms stimulus duration, super maximal voltage). At optimal length, we tetanically stimulated the muscle at 100 Hz with 90 s of recovery time between stimulations (super maximal voltage, 0.5-ms pulses, and tetanic train duration of 500 ms). Tetanic stimulations were repeated during biaxial loading of the muscle sheet. Biaxial loading was achieved by adjusting the muscle to optimal length, and then muscle was stretched in the direction transverse to the muscle fibers by either 1 or 2 g. Muscle was then maximally stimulated at 100 Hz, and this sequence of stimulations during uniaxial and biaxial loading was repeated three times. Figure 1 demonstrates how the diaphragm muscle was subjected to a biaxial load before the muscle was tetanically stimulated. To acquire force frequency curves, a separate set of experiments using six 129/SvJ normal mice (weight: 20.32 ± 3.72 g; age: 29 ± 3 days) and seven ASG-null mice (weight: 17.59 ± 1.54; age: 30 ± 4 days) was conducted. The diaphragm muscle was stimulated at 10, 30, 50, 60, and 100 Hz (super maximal voltage, 0.5-ms pulses, and tetanic train duration of 500 ms) with 120 s of recovery time between any two consecutive tetanic stimulations. Two additional force frequency data were acquired during biaxial loading of the muscle in the presence of either 1 or 2 g of muscle force applied in the direction transverse to the muscle fibers. All data for the contractile properties protocols were acquired at 300 Hz using a data acquisition board (model Lab-PC-1200/AI, National Instruments) and LabVIEWsoftware (version 4.0) applied in the transverse direction to the long axis of the muscle fibers.


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Fig. 1.   Schematic showing how the diaphragm muscle was subjected to biaxial loading during maximal tetanic stimulation. The muscle was first adjusted to optimal length (A). Transverse force of either 1 or 2 g was applied in the transverse direction to the muscle fibers (B). Muscle is under a biaxial load (C).

Statistical analysis. Statistical differences between groups were assessed by ANOVA with use of the SAS Procedure "Mixed" Program. The model was a two-factor fixed or random effects model for two groups (ASG-null vs. controls) and two treatments (uniaxial vs. biaxial). A P value of 0.05 was chosen as the acceptable level of significance throughout the analysis of all data.

Thickness measurements. Unstressed muscle thickness measurements were obtained from the excised muscles, a digital image of the muscle surface was generated, and surface area was determined by using Image Tool (version 2.0, http://ddsdx.uthscsa.edu). Excess water was removed from the surface of the tissue with a cotton-tipped swab, and the tissue sample was immediately weighed. Thickness was computed as t = m/Ad, where t is muscle thickness in centimeters, m is muscle mass in grams, A is the surface area of the sample in centimeters squared, and d is the density of the muscle and is equal to 1.06 g/cm3 (5).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Stiffness is increased in the ASG -/- diaphragm. Data in Fig. 2 show representative length-tension curves for the ASG -/- 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 ASG -/- and controls exhibited hysteresis. That is, at the same tension, the muscle exhibited lower mechanical strain on loading than on unloading. It appears that hysteresis is smaller in the ASG -/- muscles compared with normal muscles. Furthermore, the length-tension curve of the normal mouse diaphragm shifts to the right relative to the length-tension curve in the transverse fiber direction. This suggests that the muscle has greater extensibility in the direction of muscle fibers than in the transverse plane. The length-tension curves of the ASG-null diaphragm appear to exhibit similar behavior. Extensibility ratios (lambda ) for all the ASG-null and normal wild-type mice were computed at a tension of 5 g/cm. In the direction along the fibers (AF) of the diaphragm muscle, lambda  is smaller in the ASG-null mice compared with control mice (ASG -/- AF: lambda  = 1.21 ± 0.07; ASG +/+ AF: lambda  = 1.45 ± 0.14; P < 0.05). In the direction transverse to muscle fibers (TF), lambda  is smaller in the ASG-null mice than in the control mice (ASG -/- TF: lambda  = 1.10 ± 0.09; ASG +/+ TF: lambda  = 1.15 ± 0.17; P < 0.05). At a tension of 5 g/cm, the axial strains are 24 and 39% for ASG -/- and control mice, respectively, yielding a compliance ratio of 0.89. A compliance ratio that is less than one implies that at the same level of applied tension the muscle is less extensible compared with its counterpart-in this case, between the muscles of the ASG -/- mice and the control mice. At the same level of tension, 5 g/cm, the transverse strains for the ASG -/- and normal wild-type mice are 10 and 15%, respectively, yielding a compliance ratio of 0.95. These data suggest that muscles lacking ASG are less extensible and less viscous than in normal mice.


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Fig. 2.   Length-tension relationships of the diaphragm for alpha -sarcoglycan (ASG) -/- and age-matched control mice. The diaphragm muscles were passively lengthened and passively shortened in the directions along and transverse to the muscle fibers. Lengthening and shortening curves in the direction along the muscle fibers are represented by the symbols open circle  and  for the control and ASG -/-, respectively, and data in the direction transverse the fibers are represented with the symbols diamond  and star . The curves for the ASG -/- mice in the directions along (AF) and transverse (TF) to the muscle fibers is shifted to the left compared with controls, although the shift is markedly greater in the AF direction. At a tension of 5 g/cm, the tensile strain in the direction of the fibers is 24 and 39% for ASG -/- and control mice, respectively, yielding a compliance ratio of about 0.9. The tensile strain at the same level of tension transverse to the muscle fibers is 10 and 15%, yielding a compliance ratio of 0.95. These data suggest that muscles lacking ASG appear to have less hysteresis and are less extensible than normal mice, uniaxially.

Data in Fig. 3 show length-tension curves during biaxial loading of the diaphragm muscle sheet from a representative ASG mutant and a control mouse. Muscles from null-mutant mice are significantly less extensible than controls (ASG -/-: lambda = 1.15 ± 0.12; ASG +/+: lambda = 1.40 ± 0.17; P < 0.05). The diaphragm muscles exhibit nonlinear behavior. Absence of ASG appears to increase the nonlinear stiffening of the muscles especially in the fiber direction. At a tension of 5 g/cm, the tensile strains in the direction along the muscle fibers are 15 and 40% in ASG -/- and control mice, respectively, yielding a compliance ratio of 0.82. Hysteresis is essentially negligible in the ASG -/- mice compared with controls. These data suggest that during biaxial loading, muscle lacking ASG is much stiffer than muscles from normal wild-type.


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Fig. 3.   Length-tension relationships during biaxial loading of the ASG -/- and control mice diaphragms. Biaxial loading was conducted by passively stretching the diaphragm in the presence of a prestress of 1 g in the direction transverse to the muscle fibers. The muscle was then passively lengthened and passively shortened in the direction of the muscle fibers. The curves with symbols open circle  and  are cycling stretching and shortening loops in the direction of muscle fibers for control and ASG -/- mice, respectively. It appears that strains induced under biaxial loading in ASG -/- diaphragms are much less than in those of age-matched controls. At a tension of 5 g/cm, the tensile strains in the direction along the muscle fibers are 15 and 40% in ASG -/- and control mice, respectively, yielding a compliance ratio of about 0.8. These data suggest that there is a marked decrease in compliance in the diaphragm that lacks ASG compared with that of a normal diaphragm. The ASG -/- diaphragm is essentially inextensible and hysteresis has vanished under biaxial loading.

Data in Fig. 4 show length-tension curves of biceps femoris in the AF direction and TF direction. The length-tension curves for the ASG -/- mice are shifted to the left when compared with controls, with a greater shift in the AF direction. At a tension of 5 g/cm, the tensile strain in the AF direction is 10 and 17% for ASG -/- and control mice, respectively, yielding a compliance ratio of 0.93. The tensile strain at the same level of tension transverse to the muscle fibers is 12 and 16%, respectively, yielding a compliance ratio of 0.96. Using all mice, we computed extension ratios (lambda ) in the AF and TF directions of the ASG -/- and controls (ASG -/-: AF lambda  = 1.12 ± 0.09, ASG +/+: AF lambda  = 1.17 ± 0.13; ASG -/-: TF lambda  = 1.12 ± 0.05, ASG +/+: TF lambda  = 1.16 ± 0.12; P < 0.05). In the direction along muscle fibers, hysteresis is greater in the control mice than in the ASG -/- mice. These data suggest that the biceps femoris muscle lacking ASG is less extensible than normal muscles.


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Fig. 4.   Length-tension relationships of the biceps femoris for ASG -/- and age-matched control mice. The muscles were passively lengthened and passively shortened in the directions along the muscle fibers and transverse to the fibers. Lengthening and shortening curves in the direction along the muscle fibers are represented by the symbols open circle  and  for the control and ASG -/- mice, respectively, and the data in the direction transverse the fibers are represented with the symbols diamond  and star . The curves for the ASG -/- mice in the directions along (AF) and transverse (TF) 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 10 and 17% for ASG -/- and control mice, respectively, yielding a compliance ratio of 0.93. The tensile strain at the same level of tension transverse to the muscle fibers is 12 and 16%, respectively, resulting in a compliance ratio of 0.96. Hysteresis is greater in the along fibers direction of the control than the ASG -/- mouse; however, hysteresis is essentially the same between the control and ASG -/- mouse in the transverse to muscle fibers curves. These data suggest that muscles deficient in ASG are less extensible than normal.

Loss of coupling between transverse passive stress and contractile muscle force in the ASG -/- mice. The data in Fig. 5 demonstrate that biaxial mechanical loading with a transverse force of 1 g increases muscle tetanic stress only in the normal diaphragm (1 g biaxial: ASG +/+: 22.04 ± 1.48 N/cm2; ASG +/+: 24.97 ± 1.19 N/cm2; P < 0.05). In contrast to normal wild-type, differences in tetanic stress in the ASG-null diaphragm between uniaxial and biaxial loading virtually vanish regardless of the magnitude of transverse force (1 g biaxial: ASG -/- 16.194 ± 0.78 N/cm2; ASG -/-: 17.12 ± 1.15 N/cm2; 2g biaxial: ASG -/- 17.26 ± 1.07 N/cm2). This demonstrates that the effect of transverse passive stress on the contractile properties in the ASG -/- mice is negligible, whereas transverse passive stress increases contractile muscle force.


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Fig. 5.   Maximum tetanic stress in normal and ASG-null diaphragms under uniaxial and biaxial loading. Data obtained at 100-Hz stimulation from 7 normal 129/SvJ mice (weight: 17.36 ± 4.66 g; age: 30 ± 13 days) and 8 ASG-null mutant 129/SvJ mice (weight: 17.56 ± 1.66 g; age: 30 ± 11 days). Under both uniaxial and biaxial loading conditions, muscle stress production is depressed in the ASG-null mutant mice (ANOVA: P < 0.05). Effect of biaxial loading on ASG-null mutant diaphragm is not significant compared with uniaxial loading, whereas normal diaphragms demonstrate enhanced muscle stress production in the presence of biaxial load with a transverse force of 1 g (ANOVA: P < 0.05).

The force frequency curves presented in Fig. 6 demonstrate that muscle tetanic stress is significantly reduced in the ASG-null mice compared with controls. The force frequency curves for the normal mice and the null mutants reach maximum force at a stimulation frequency of about 30 Hz. Compared with normal wild-type mice, muscle force appears depressed in the ASG-null diaphragms. In addition, in normal mice muscle force per cross-sectional area during biaxial loading is greater than that during uniaxial loading, whereas in ASG-null diaphragms muscle force during uniaxial appears similar to that during biaxial loading conditions.


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Fig. 6.   Force-frequency curves for normal and ASG-null mutant mice under uniaxial and biaxial loading conditions. The open symbols are for normal wild-type mice, and the filled symbols are for ASG -/-. The triangles represent data during uniaxial loading. The squares and diamonds are data during biaxial with transverse passive force of 1 and 2 g, respectively. Data are obtained from 6 normal 129/SvJ mice (weight 20.32 ± 3.72 g; age: 29 ± 3 days) and 7 ASG -/- mice (weight 17.59 ± 1.54 g; age 30 ± 4 days). Muscle tetanic force is depressed in ASG-null diaphragms compared with normal diaphragms. Additionally, in normal mice, muscle force during biaxial loading is greater than during uniaxial loading, whereas in ASG-null diaphragms, muscle force appears similar between uniaxial and biaxial loads.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In this study, we found that muscle extensibility is decreased in skeletal muscles from the ASG-null mice. We also found that muscle force-generating capacity is depressed in the diaphragm muscle of these mice. Furthermore, we found that passive transverse stress has no effect on the contractile properties of the diaphragm in ASG-null mice, whereas muscle force is increased in the presence of transverse stress in the normal mouse.

The mouse model used in our study was developed by Liu and Engvall (17) and essentially follows the autosomal recessive LGMD exhibited by humans with a primary sarcoglycan gene defect. Analogous to this mouse model, Duclos et al. (10) developed an ASG-null mutant mouse that exhibited matching trends in complex formation and localization of the sarcoglycan complex. Both models of the null mutant ASG exhibited histopathological features of autosomal LGMD about 1 wk after birth with ongoing necrosis until the age of 9 mo. Histological and immunofluorescence analysis of skeletal muscles in 2-mo-old mice demonstrated the established sarcoglycan complex in the control mice and the degeneration of the beta -sarcoglycan in the ASG-null mice. This deficiency is also noticed in the delta - and gamma -sarcoglycans, leading to a disruption in the sarcoglycan complex (17).

The passive and maximally stimulated uniaxial length-tension curves of the diaphragm muscle have been measured (11, 12, 16, 18-22). The length-tension relationship of a homogeneous elastic material is stiffer when subjected to a biaxial than when subjected to a uniaxial load. This is an added complexity in relating the length-tension relationship measured in isolated diaphragm preparations to in vivo measurements. Limited data are available on the diaphragm muscle properties under biaxial loading (4, 5, 13), and the relationship between stress in the transverse direction and the length-tension properties of the muscle in the direction of the fibers is unknown. We measured the longitudinal and transverse strains in the excised (4) and intact (5) diaphragms. Data from these studies have shown that the diaphragm muscle stiffness properties are anisotropic, with a greater stiffness in the transverse fiber direction than that in the fiber direction. If the diaphragm were extensible in the direction transverse to muscle fibers, then during active contraction, as transdiaphragmatic pressure and stress in both directions increase, the diaphragm would expand in the transverse direction as it contracts along the muscle fibers direction (6).

We recently investigated the mechanical role of desmin (3), a cytoskeletal protein in muscles. We demonstrated that desmin integrates the three-dimensional properties of skeletal muscles by coupling the longitudinal and transverse properties of the diaphragm. In our current study, we demonstrated that transverse loads increase muscle maximal contractile force production in the diaphragm, indicating the presence of structures that couple longitudinal and transverse properties in the diaphragmatic muscle. Our data suggest that desmin may not be the only structural protein that integrates the longitudinal and transverse elements in skeletal muscles. The sarcoglycan complex may be another structural element that could transmit muscle force between the longitudinal and transverse elements. ASG is one of the many components of the dystrophin-associated complex, which could function at least in part to transmit forces generated by the sarcomeric proteins across the cell membrane. This group of membrane-associated structural proteins is found to be highly concentrated at the ends of muscle fibers at the muscle tendonous junction where contractile muscle forces and passive forces would be transmitted across the cell membrane (3).

In contrast to desmin-null mice, which show more pronounced muscle extensibility in the transverse plane to the fibers, skeletal muscles from the ASG-null mice are stiffer in the axial, as well as in the transverse, direction to the muscle fibers compared with muscles from age-matched normal wild-type mice. Our findings are in agreement with those published by Duclos et al. (10) on hindlimb muscles. Although data were not presented, the investigators (10) stated that their data on passive stretch of the extensor digitorum longus and soleus (EDL) muscles demonstrated an increase in the resistance to passive stretch in the ASG-null muscles compared with controls.

Reduced muscle compliance has generally attributed to collagen accumulation (2, 14, 23). However, there are other studies that have demonstrated no correlation between the proportion of collagen and passive stiffness in striated muscles. In particular, in the rat soleus muscle, the increased collagen content during ageing is not associated with changes in muscle stiffness (1). Furthermore, diaphragm stiffness is decreased in the cardiomyopathic Syrian hamster, whereas the surface area of collagen was increased in those animals (8). The ASG-null mice develop muscle necrosis on day 7 after birth, and according to the data by Duclos et al. (10), these mice show ongoing muscle necrosis with increasing age. Our experiments were conduced on mice that are about 30 days old. Therefore, we cannot rule out the possibility that the sarcoglycan complex is a load-bearing element in skeletal muscles despite the increased stiffness in the ASG-null mice.

We chose the biceps femoris because in vivo it experiences mechanical loading only along the length of the muscle fibers, whereas the diaphragm experiences mechanical loads not only along the muscle fibers but also transverse to the muscle fibers. Furthermore, the biceps femoris muscle fibers run along the length of the muscle, whereas muscle fibers in the EDL and soleus muscles are oriented at an angle to the long axis of the muscle. Therefore, it is easier to apply a transverse load to the biceps femoris than to either the EDL or soleus muscles. Furthermore, the biceps femoris is a flat muscle, and, therefore, it is easy to apply mechanical stretch in the plane of the muscle sheet.

The increased muscle stiffness in muscle fibers of the diaphragm during uniaxial loading is demonstrated in data shown in Fig. 2. The significant shift to the left of the length tension curves of the diaphragms of ASG -/- mice compared with controls demonstrate reduced muscle extensibility in the directions of the fibers, as well as in the direction transverse to the fibers. Similar trends are seen in Fig. 3 where the muscle length-tension relationships during biaxial loading of the ASG -/- diaphragm demonstrate a very significant increase in stiffness compared with the controls. The extensibility and muscle compliance of the diaphragm in the ASG -/- is significantly smaller during biaxial lengthening than during uniaxial lengthening in the muscle fiber direction. Differences between passive lengthening and passive shortening data suggest that the muscles exhibit viscoelastic behavior. Our data suggest that hysteresis is smaller in the ASG -/- compared with controls. This suggests that the ASG complex may contribute to the viscoelastic properties of the muscles. In particular, it may serve as an energy-dissipating complex, at least during passive stretching. The effect of ASG deficiency on muscle stiffness is not specific to the diaphragm; the biceps femoris muscles are less extensible in the ASG -/- mice. Furthermore, the effect of ASG deficiency in altering muscle extensibility and stiffness is more pronounced in the fiber direction than in the transverse fiber direction (Fig. 4). These data are consistent with a possible mechanical role of the sarcoglycan complex in modulating passive stiffness in skeletal muscles.

Data in Figs. 5 and 6 suggest that the sarcolemma of the diaphragm is not capable of transmitting muscle force in the direction transverse to fibers in the ASG mutant mice. This determines a possible role of ASG complex in transmitting muscle force between the longitudinal and transverse elements. ASG appears to contribute to the coupling of the transverse and longitudinal mechanical properties of the diaphragm muscles through structural elements. These results are similar with those reported by us (3) in which muscle force production was not altered during biaxial loading compared with that during uniaxial loading in desmin-deficient mice.

Our data suggest that the sarcoglycan complex may serve a complex mechanical function in the diaphragm by contributing to muscle stiffness, muscle viscoelasticity, and the modulation of the contractile properties of the muscle.


    ACKNOWLEDGEMENTS

We thank Dr. Eva Engvall for providing the ASG -/- mouse model, conducting the immunocytochemical labeling analysis, and providing discussion of the data.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute grant HL-63134.

Address for reprint requests and other correspondence: A. M. Boriek, Baylor College of Medicine, One Baylor Plaza, Dept. of Medicine, Pulmonary Section, 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.

10.1152/ajpcell.00326.2002

Received 12 July 2002; accepted in final form 27 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Alnaqeeb, MA, ALZaid NS, and Goldspink G. Connective tissue changes and physical properties of developing and ageing skeletal muscle. J Anat 139: 677-689, 1984[ISI][Medline].

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