Journal of Cell Science 107, 1477-1483 (1994) 1477 Printed in Great Britain © The Company of Biologists Limited 1994

Talin, and DRP () concentrations are increased at mdx myotendinous junctions following onset of necrosis

Douglas J. Law1,*, David L. Allen1 and James G. Tidball1,2 1Department of Physiological Science and 2Jerry Lewis Neuromuscular Research Center, University of California, 405 Hilgard Avenue, Los Angeles, CA 90024-1527, USA *Author for correspondence at present address: School of Biological Sciences, University of Missouri, 5100 Rockhill Rd, Kansas City, MO 64110-2499, USA

SUMMARY

Duchenne muscular dystrophy (DMD) and the myopathy thin filament-membrane interactions at myotendinous seen in the mdx mouse both result from absence of the junctions. Blots also showed an increase (143%) in the dys- . Structural similarities between dys- trophin-related protein called utrophin, another myotendi- trophin and other cytoskeletal , its enrichment at nous junction constituent, which may be able to substitute myotendinous junctions, and its indirect association with for dystrophin directly. Muscle samples from 2-week-old laminin mediated by a transmembrane glycoprotein animals, a period immediately preceding the onset of complex suggest that one of dystrophin’s functions in muscle necrosis, showed no significant differences in normal muscle is to form one of the links between the protein concentration between mdx and controls. Quanti- and the . Unlike tative analyses of confocal images of myotendinous Duchenne muscular dystrophy patients, mdx mice suffer junctions from mdx and control muscles show significantly only transient muscle necrosis, and are able to regenerate higher concentrations of and vinculin at the myotendi- damaged muscle tissue. The present study tests the hypoth- nous junctions of mdx muscle. These findings indicate that esis that mdx mice partially compensate for dystrophin’s mdx mice may compensate in part for the absence of dys- absence by upregulating one or more dystrophin-indepen- trophin by increased expression of other molecules that dent mechanisms of cytoskeleton-membrane association. subsume dystrophin’s mechanical function. Quantitative analysis of immunoblots of adult mdx muscle samples showed an increase of approximately 200% for Key words: dystrophin, myotendinous junction, talin, vinculin, vinculin and talin, cytoskeletal proteins that mediate utrophin

INTRODUCTION in DMD, mdx muscle suffers only a transient period of degen- eration at 3-4 weeks of age, followed by nearly complete Dystrophin, the protein absent in Duchenne muscular regeneration and restoration of function (Anderson et al., 1988; dystrophy (DMD) (Hoffman et al., 1987a), is thought to Coulton et al., 1988; Cullen and Jaros, 1988). This regenera- function as a structural link between the muscle cell cytoskele- tion suggests that mdx mice are able to compensate for dys- ton and the extracellular matrix. This expectation is supported trophin’s absence, possibly by upregulating the expression of by the structural similarities between dystrophin and the one or more proteins that can function similarly to dystrophin. cytoskeletal proteins α- and (Byers et al., 1989; For example, the dystrophin-related protein (DRP) utrophin Hammonds, 1987; Koenig et al., 1988). Also, dystrophin is (Love et al., 1989), an autosomal gene product that is located subjacent to the muscle (Arahata et al., extremely similar in primary structure to dystrophin (Tinsley 1988; Cullen et al., 1990; Watkins et al., 1988), and biochem- et al., 1992), can bind to the DAG complex and may substitute ical studies have shown binding affinity between dystrophin’s for dystrophin in some mdx tissue (Matsumura et al., 1992). N-terminal domain and actin (Hemmings et al., 1992; Way et We propose the alternative hypothesis that regeneration of al., 1992), and between part of its C-terminal domain and a adult mdx muscle is associated with upregulation of proteins transmembrane glycoprotein complex (Suzuki et al., 1992). that form transmembrane assemblies that normally function in One subunit of that dystrophin-associated glycoprotein (DAG) parallel with dystrophin. One such parallel assembly comprises complex can bind to the basement membrane component vinculin, talin, , and one of several integrin-binding laminin (Ervasti and Campbell, 1993; Ibraghimov-Beskrov- extracellular matrix proteins (Burridge and Mangeat, 1984; naya et al., 1992), thereby completing the transmembrane asso- Horwitz et al., 1986; Hynes, 1987). The DAG-dystrophin ciation of structural proteins. based assembly and integrin-talin based assembly are similar Dystrophin is also absent in the mdx mouse (Hoffman et al., in that they are both concentrated at myotendinous junctions 1987). However, unlike the progressive muscle wasting seen (MTJs), the principal sites of force transmission across the 1478 D. J. Law, D. L. Allen and J. G. Tidball muscle cell membrane (Bozyczko et al., 1989; Byers et al., values for mdx and control muscles were compared for each blot using 1991; Samitt and Bonilla, 1990; Shear and Bloch, 1985; Student’s t-test. Shimizu et al., 1989; Swasdison and Mayne, 1989; Tidball et al., 1986), and they both form links between the actin Immunofluorescence microscopy cytoskeleton and extracellular structural proteins. The present The tibialis anterior muscles that were not used to make samples for study tests the upregulation of this parallel mechanism for SDS-PAGE were dissected out of mdx and control animals, frozen in liquid isopentane cooled in liquid nitrogen, and stored at −80¡C in cytoskeleton-membrane association by answering the isopentane. Samples were warmed to −20¡C in a cryostat, sectioned following questions: (1) does adult mdx muscle have more longitudinally at 10 µm thickness, and sections mounted on gelatin- vinculin and talin than control muscle? (2) if so, is the increase coated slides. Sections were blocked for 1 hour in buffer A with 0.2% in protein localized to the MTJ? and (3) if vinculin and talin gelatin, 0.05% Tween-20, and 0.3% nonfat dry milk, rinsed twice for are elevated, is this a general feature of mdx muscle at all ages 2 minutes in buffer A, then incubated for 3 hours in anti-vinculin or or rather a change observed only following the cellular damage anti-talin, diluted 1:20 in buffer A. Sections were rinsed 2× 2 minutes that results from dystrophin’s absence? in buffer A, incubated for 30 minutes in the above blocking buffer, then rinsed twice more in buffer A. The sections were then incubated for 1 hour in TRITC-conjugated goat anti-mouse IgG (Southern Biotechnology, Birmingham, AL) diluted 1:250 in buffer A, then MATERIALS AND METHODS rinsed 3× 5 minutes in buffer A. Finally, sections were rinsed for 2 minutes in distilled water, mounted with aqueous medium, and pho- Antibodies tographed using an Olympus BH-2 microscope equipped with epiflu- Monoclonal antibodies to vinculin and talin were purchased from orescence and Nomarski differential interference contrast. Controls Sigma (anti-human vinculin, clone hVIN-1; anti-chicken talin, clone consisted of sections processed identically, except buffer A was used 8d4). Polyclonal antiserum made in rabbit against a TrpE fusion in place of primary antibody. protein consisting of the C-terminal domain of the dystrophin-related The relative amounts of vinculin and talin present at myotendinous protein (DRP) was a gift from Dr Tejvir Khurana (Khurana et al., junctions were estimated using digital analysis of confocal images of 1990). Affinity-purified polyclonal rabbit anti α-actinin, made the above immunolabeled sections of mdx and control muscle. against purified chicken gizzard α-actinin, was a gift from Dr Keith Sections were examined briefly using a Nikon Optiphot microscope Burridge. equipped with conventional epifluorescence, to identify fields that included labeled MTJs. Confocal images of the MTJs were obtained Immunoblots with a Molecular Dynamics (Sunnyvale, CA) laser confocal micro- Tibialis anterior muscles from one hindlimb of adult (11-month) or scope, using a ×60 oil-immersion objective with a numerical aperture 2-week-old mdx and C57BL/10SnJ control mice were dissected out of 1.4. Serial images of 1.0 µm thickness were examined, and the fresh, and frozen immediately in liquid nitrogen. These ages were image halfway between the top and bottom of the labeled MTJ was selected for analysis because they represent stages in the mdx patho- chosen for quantitative analysis. This was done to eliminate possible physiology before the onset of muscle necrosis and inflammation (2 variation in signal intensity due to the inclusion of images of the top weeks) or following complete regeneration of the muscle (11 months). or bottom surfaces of a muscle fiber. Three transects were drawn Muscles undergoing necrosis were not analyzed because they contain across the labeled MTJ interface, from the extracellular space into the a large volume fraction of inflammatory cells and necrotic fibers at interior of the muscle cell. The intensity of the fluorescent signal was that stage. C57BL/10SnJ was used as control because it is the strain measured at each pixel along a given transect, and the peak values from which mdx mutants were derived. The mdx mutation is a point were recorded. In addition, the intensity value at the intracellular end mutation in the dystrophin gene (Sicinski et al., 1989) and is unknown of each transect was recorded, to estimate background fluorescence to affect any other gene. Samples were homogenized in SDS-PAGE intensity. Peak intensity values and background values were obtained sample buffer (Laemmli, 1970) without Bromphenol Blue, and for the MTJs of six muscle cells from each of three mdx mice, and an soluble protein concentrations determined by UV spectrophotometry. equal number of values obtained from three C57 control mice. This Gels were loaded with 50 µg total protein per lane, and samples yielded a total of 54 peak values from mdx MTJs, and the same separated electrophoretically. Proteins were transferred to nitrocellu- number from controls. All settings, including laser wattage and pho- lose (Burnette, 1981), and the transfer gels stained with Coomassie tomultiplier tube voltage, were identical for all specimens. Means of Blue to ensure uniform transfer. In addition, uniformity of sample the peak values and the background values from the two groups of loading was checked on identically-loaded gels that were subse- animals were compared using Student’s t-test. Statistical significance quently stained with Coomassie Blue. Nitrocellulose blots were was assigned to differences in means for which P<0.05. blocked for 2-14 hours in 50 mM Tris, pH 7.5, 150 mM NaCl, and 0.1% NaN3 (buffer A), with the addition of 0.2% gelatin, 0.05% Tween-20, and 3% nonfat dry milk. Blots were then overlaid for 90 minutes with antisera diluted in buffer A, as follows: anti-vinculin, RESULTS 1:200; anti-talin, 1:40; anti-DRP, 1:250; anti α-actinin, 1:20. Blots were washed 6× 10 minutes in the above blocking buffer with the The specificities of the antibodies used in the present study are nonfat dry milk concentration reduced to 0.3%, and then overlaid 60- shown on western blots of normal adult mouse 90 minutes with alkaline phosphatase-conjugated, species-specific samples (Fig. 1). The anti-vinculin labeled both the 117 kDa second antibodies (Sigma, St Louis, MO) diluted 1:2000 in buffer A vinculin band, and a 135 kDa band that has been previously with 0.2% gelatin, 0.05% Tween-20, and 5% inactivated horse serum. × identified as meta-vinculin, a vinculin-related protein (Byrne After 6 10 minute washes, bound antibodies were detected by incu- et al., 1992). Anti-talin labeled a single band at 235 kDa, and bating the blots with 0.3 mg/ml nitro blue tetrazolium and 0.15 mg/ml anti-α-actinin a single band at 100 kDa. The anti-DRP labeled 5-bromo-4-chloro-3-indolyl phosphate in 0.1 M NaHCO3, 1.0 mM MgCl2, pH 9.8. Blots were quantified using a laser densitometer a doublet in the control muscle sample at approximately 400 (Ultroscan, Pharmacia, Piscataway, NJ). Samples from 3 mdx and 3 kDa (Fig. 1b). The stronger, lower band is dystrophin, with control animals were run on the same gel. Integrated absorbencies for which this polyclonal antiserum cross-reacts (Khurana et al., the immunostained bands in each lane were determined, and mean 1991), and the weaker, higher band is the DRP, which has been

Increased talin, vinculin and DRP in mdx 1479

Fig. 2. Comparison of structural protein concentrations in mdx and control mouse skeletal muscle. Top: 8% gel, stained with Coomassie Blue, of skeletal muscle Fig. 1. Antibody specificity, shown by immunoblotting. (A) SDS- samples from three PAGE and immunoblots of control mouse skeletal muscle separated mdx (D) and three on an 8% acrylamide gel. Lane 1, Coomassie Blue-stained gel. control (C) mice. Positions of molecular mass standards are marked: 200 kDa, ; Below: immunoblots 116 kDa, carbonic anhydrase; 97.5 kDa, phosphorylase; 68 kDa, show an elevation in BSA; 42 kDa, ovalbumin. Lane 2, immunoblot with anti-vinculin. concentration in the Lower band at 117 kDa is vinculin; upper band is metavinculin. Lane mdx samples for 3, immunoblot with anti-talin. Lane 4, immunoblot with anti α- vinculin (vn), talin (tn), and DRP (drp). actinin. (B) Control mouse skeletal muscle sample separated on a 5% α acrylamide gel, overrun to maximize resolution of high molecular No difference in - mass bands. Lane 1, Coomassie Blue gel shows 200 kDa myosin actinin (aa) heavy chain at the bottom of the gel. Lane 2, immunoblot with anti- concentration was DRP. A pair of closely-spaced bands at approximately 400 kDa is detected. labeled (arrows); the upper band is DRP, and the lower is dystrophin, with which this antiserum cross-reacts. The faint band at lower molecular mass is likely a breakdown product of dystrophin. differences in concentration of any of the proteins from the 2- week mice. Frozen sections of adult mdx and control mouse hindlimb shown to migrate slightly slower than dystrophin in SDS- muscle were immunolabeled for talin and vinculin to determine PAGE (Ohlendieck et al., 1991). if the increased amounts of these proteins seen in immunoblots Blots of skeletal muscle samples from adult mdx and control were localized at the MTJ. The results of conventional fluo- animals (Fig. 2) showed increased concentrations of vinculin, rescence microscopy showed that MTJs were the most talin, and DRP in the mdx muscle relative to control levels. numerous and most intensely labeled structures for talin and There was no apparent difference in the concentration of α- vinculin in both C57 and mdx muscle (Figs 3 and 4). Sections actinin in the adult mdx samples relative to controls, indicat- processed by identical labeling procedures but from which ing that the elevations in the other three proteins are specific, primary antibodies were eliminated showed no MTJ labeling. and not due to a more general upregulation of cytoskeletal Measurements of maximum fluorescence intensity at MTJs in protein expression in mdx muscle. Blots of muscle samples single confocal images demonstrated a 42% increase in from 2-week-old mice showed no systematic differences intensity in mdx muscles labeled for vinculin, and a 36% between mdx and controls, regarding concentration of any of increase in talin, compared with the values obtained from the above proteins (not shown). These observations were control muscles (Table 2). The mean values for background confirmed by quantitative comparisons of the immunoblots of fluorescence intensity from these samples were not signifi- 2-week and 11-month muscles, using scanning densitometry. cantly different, indicating that the differences in maximum The results (Table 1) indicate statistically-significant increases MTJ signal are due to differences in the amount of antigen in the concentrations of vinculin (170%), meta-vinculin present in the confocal section, rather than to a non-specific (311%), talin (218%), and DRP (143%) in muscles from adult increase in immunoreactivity of the mdx tissue compared to mdx mice relative to control concentrations, and no significant controls. The difference in the magnitude of the increases seen

Table 1. Protein concentration, relative to 100% of values Table 2. Fluorescence intensity at MTJs in single confocal from C57 control samples (mean ± s.e.m.) sections (mean ± s.e.m.; arbitrary units) Vinculin Meta-vinculin Talin DRP α-actinin mdx Imax C57 Imax mdx I0 C57 I0 2-week C57 100±19 100±37 100±6 100±21 100±7 Anti-vinculin 142*±6 100±8 85±9 100±8 2-week mdx 86±14 85±26 82±10 107±13 94±10 Anti-talin 136*±4 100±4 100±7 100±14 Adult C57 100±18 100±44 100±18 100±14 100±10 Adult mdx 270*±5 411*±8 318*±17 243*±12 104±2 Imax = maximum intensity. I0 = background intensity. *P < 0.05. n = 54. n = 3. *P < 0.05. 1480 D. J. Law, D. L. Allen and J. G. Tidball

Fig. 4. Longitudinal sections of C57 and mdx muscle labeled with anti-vinculin. (A) C57 muscle. (B) Mdx muscle. Arrowheads indicate MTJ. Bar, 50 µm.

defect that gives rise to muscle fiber damage, characterized in early pathological stages by Z-band streaming, dilation of sar- coplasmic reticulum and myofibril disorganization (Cullen and Jaros, 1988; Torres and Duchen, 1987). This structural damage has been related to an increase in intracellular calcium con- centrations resulting from calcium entry through membrane tears (Mokri and Engel, 1975) or leaky calcium channels (Turner et al., 1993). Supranormal calcium concentrations are expected to activate intracellular calcium-dependent (), which can then initiate much of the degenerative Fig. 3. Frozen longitudinal sections of C57 and mdx muscle labeled processes observed in dystrophin-deficient muscle (Boden- with anti-talin. (A) Nomarski optics image of C57 muscle. MTJ is steiner and Engel, 1978; Gorospe and Hoffman, 1992). If this outlined by arrowheads. Tendon (T) lies in lower portion of general mechanism associating dystrophin deficiency with micrograph. (B) Same field as shown in A, except viewed by indirect muscle cell death is true, then two possible hypotheses can be immunofluorescence following anti-talin labeling. (C) Mdx muscle proposed for the mechanism by which mdx mice are able to labeled with anti-talin. MTJs (arrowheads) are indicated. Bar, 50 µm. recover from the onset of muscle necrosis: (1) mdx muscle compensates for dystrophin by increased expression of analogous proteins; or (2) mdx muscle arrests necrosis by con- in the confocal data compared to those from densitometry of trolling -mediated proteolysis. the blots likely resulted from differences in the conditions of Results of previous studies (Man et al., 1991; Matsumura et binding between antibodies and antigens in the two methods, al., 1992) have supported the first hypothesis by showing that and thus cannot be interpreted. DRP, a protein that is structurally similar to dystrophin, is present at elevated concentrations in mdx muscle. Strong DISCUSSION evidence that DRP can ‘rescue’ dystrophin-deficient muscle fibers comes from Matsumura et al. (1992), who showed that Dystrophin deficiency in mdx and DMD muscle is the primary the increase in DRP concentration in cardiac and small-caliber Increased talin, vinculin and DRP in mdx 1481 skeletal muscle fibers is accompanied by near-normal levels of cytoskeleton associations with the membrane in dystrophic expression of the dystrophin-associated glycoproteins (DAG). muscle, without compensating for dystrophin deficiency However, the increase in DRP seen in quadriceps muscles did causing increased intracellular calcium concentration. Thus, not result in a similar preservation of control levels of DAG any pathology resulting from elevated calcium concentration expression. Instead, as demonstrated in previous studies in dystrophic muscle must be controlled by an additional (Ervasti et al., 1990; Ohlendieck and Campbell, 1991), the mechanism. DAG concentration was greatly reduced in the mdx hindlimb Recent investigations that have examined potential mecha- muscles. If the normal function of dystrophin requires its inter- nisms by which calcium-dependent proteolysis in dystrophin- action with the DAGs, then the increase in DRP concentration deficient muscle can be controlled have focused on two possi- in mdx muscle shown in the previous (Man et al., 1991; bilities: (1) calcium may be buffered in mdx muscle (Gailly et Matsumura et al., 1992) and current studies may be sufficient al., 1993); or (2) calpains may be inhibited (Spencer and to compensate for dystrophin’s absence in the small-caliber Tidball, 1992). Current evidence offers some support for and fibers in mdx mice (Matsumura et al., elevated calcium buffering by parvalbumin, which is expressed 1992), but not in other mdx skeletal muscles. at higher levels in mdx muscle than in controls (Gailly et al., We propose that the increased concentration of vinculin and 1993). In spite of this, parvalbumin concentrations remain talin, demonstrated here, also may be a mechanism by which lower in mdx muscle (Sano et al., 1990), so the role of parval- mdx mice can compensate for the absence of dystrophin. bumin in mdx pathology remains unclear. Other calcium- Vinculin and talin are well-characterized components of the binding proteins, such as calmodulin, may be more important that anchors actin-containing stress fibers to in this Ca2+ buffering role. Calmodulin concentrations are the cell membrane at focal adhesions of cultured fibroblasts elevated in dystrophic hamster muscle (Klamut et al., 1987), (Beckerle and Yeh, 1990; Otto, 1990). Vinculin associates although this dystrophy does not result from dystrophin defi- indirectly with actin, probably via the actin-binding protein ciency. Calmodulin levels have not yet been analyzed in mdx tensin (Bockholt et al., 1992; Davis et al., 1991). Vinculin also muscle. binds to talin (Burridge and Mangeat, 1984), which in The possibility that calcium-dependent necrosis may be con- associates with members of the integrin family of extracellu- trolled by regulating calpain activity has been supported by lar matrix receptors (Horwitz et al., 1986). Thus, this observations that leupeptin, an inhibitor of calpain and other mechanism is analogous to the dystrophin-based mechanism, thiol proteases, can inhibit proteolysis in mdx muscle (Turner in that they both provide links between the actin cytoskeleton et al., 1993). Furthermore, recent measurements of calpain con- and extracellular matrix. Both mechanisms are normally centration and activity in adult mdx and control muscle show present at MTJs (Samitt and Bonilla, 1990; Shear and Bloch, that although calpain is present in higher concentrations in mdx 1985; Shimizu et al., 1989; Tidball et al., 1986), so it is likely muscle, the net Ca2+-dependent proteolytic activity of mdx that they function in parallel in normal muscle. The two mech- muscle extracts is lower than in controls (Spencer and Tidball, anisms may not be completely independent, as indicated by a 1992). Thus, inhibition of calpain activity may be an additional recent study showing binding affinity between dystrophin and means by which mdx muscle arrests the necrotic process. talin in vitro (Senter et al., 1993). However, in the absence of We conclude that mdx muscle can compensate for some dystrophin, talin and vinculin could retain the ability to link structural defects associated with dystrophin deficiency by the actin cytoskeleton with the membrane, through . increasing the concentration of proteins that serve analogous Our results showing no difference in vinculin or talin con- functions in mediating cytoskeletal-membrane associations. centrations in muscles from 2-week-old mice indicate that the However, these analogous proteins are not capable of compen- vinculin-talin mechanism has not yet been upregulated at this sating for the defect that gives rise to increased intracellular stage in the mouse’s life. This indicates that the compensation calcium. Further studies of the functional domains of dystrophin is not a direct response to the absence of dystrophin, because may help determine whether the region of the molecule that dystrophin is also absent in those young animals, and shows mediates cytoskeletal-membrane associations is distinct from that the increased concentration of talin and vinculin is not con- that involved in control of intracellular calcium concentrations. stituitive in mdx, but rather is a condition that develops later in mdx pathology. It may be a response either to the cellular injury This investigation was supported by the Louis and Emma Benzak that begins at about 3 weeks, or to part of the process of muscle Neuromuscular Research Fellowship (D.J.L.) and grants from the regeneration. It is also noteworthy that this compensatory National Institutes of Health (AR 40343) and Muscular Dystrophy mechanism does not exist in DMD muscle. 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