Distribution of ten laminin chains in dystrophic and regenerating muscles

Neuromuscular Disorders 9 (1999) 423±433 www.elsevier.com/locate/nmd

Distribution of ten laminin chains in dystrophic and regenerating muscles Bruce L. Patton a, Anne M. Connolly b, c, Paul T. Martin a, Jeanette M. Cunningham a, Shobhna Mehta b, Alan Pestronk b, Jeffrey H. Miner d, Joshua R. Sanes a,* a

Department of Anatomy and Neurobiology, Washington University Medical Center, 660 South Euclid Avenue, St. Louis, MO, 63110, USA b Department of Neurology, Washington University Medical Center, 660 South Euclid Avenue, St. Louis, MO, 63110, USA c Department of Pediatrics, St. Louis Children's Hospital, Washington University Medical Center, 660 South Euclid Avenue, St. Louis, MO, 63110, USA d Department of Medicine, Washington University Medical Center, 660 South Euclid Avenue, St. Louis, MO, 63110, USA Received 20 November 1998; received in revised form 22 February 1999; accepted 7 March 1999

Abstract Using immunohistochemical methods, we assessed the distribution of all 10 known laminin chains (a 1-5, b 1-3, g 1 and g 2) in skeletal muscles from patients with Duchenne, congenital, limb girdle, or Emery±Dreifuss muscular dystrophies. The a 2, b 1 and g 1 chains were abundant in the basal lamina surrounding muscle ®bers in normal controls; a 1, a 3±a 5, b 3, and g 2 were undetectable; and b 2 was present at a low level. Compared to controls, levels of the a 5 chain were increased in muscles from many dystrophic patients; levels of b 1 were reduced and/or levels of b 2 were increased in a minority. However, these changes were neither speci®c for, nor consistent within, diagnostic categories. In contrast, levels of a 4 were increased in muscles from all patients with a 2 laminin (merosin)-de®cient congenital muscular dystrophy. Loss of a 2 laminin in congenital dystrophy is disease-speci®c but some other changes in laminin isoform expression in dystrophic muscles could be secondary consequences of myopathy, denervation, regeneration or immaturity. To distinguish among these possibilities, we compared the laminins of embryonic, denervated, regenerating, and mutant mouse muscles with those in normal adult muscle. Embryonic muscle basal lamina contained a 4 and a 5 along with a 2, and regenerating muscle re-expressed a 5 but not a 4. Levels of a 5 but not a 4 were increased in dystrophin (mdx) mutants and in dystrophin/utrophin double mutants (mdx:utrn 2 / 2 ), models for Duchenne dystrophy. In contrast, laminin a 4 was upregulated more than a 5 in muscles of laminin a 2 mutant mice (dy/dy; a model for a 2-de®cient congenital dystrophy). Based on these results, we hypothesize that the expression of a 5 in many dystrophies re¯ects the regenerative process, whereas the selective expression of a 4 in a 2-de®cient muscle is a speci®c compensatory response to loss of a 2. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Laminin chains, Distribution, Dystrophic muscle; Regenerating muscle

1. Introduction Each muscle ®ber in skeletal muscles is surrounded by a layer of extracellular matrix material called the basal lamina (BL). Major components of all BLs are the laminins, large heterotrimeric glycoproteins composed of a , b , and g chains. To date, 5 a , 3b , and 2 g chains have been described, which assemble into at least 11 distinct heterotrimers [1,2]. The major laminin of muscle ®ber BL is a 2/ b 1/g 1 [3±5], which is called laminin 2 [6]. Several sets of studies implicate laminins in the pathogenesis of muscular dystrophy. Firstly, BLs serve as scaffolds to * Corresponding author. Tel.: 11-314-362-2507; fax: 11-314-7471150. E-mail address: [email protected] (J.R. Sanes)

organize and orient muscle regeneration [7]. Laminin 2 is likely to be critical for this function not only because it is a major structural component of muscle BL, but also because it has potent myogenic activity [8]. Secondly, laminin 2 is associated with a set of transmembrane and intracellular proteins, the dystrophin-associated glycoprotein complex (DGC), that has been implicated in several muscular dystrophies [9]. Dystroglycans, which are membrane-associated DGC components, bind laminin 2 extracellularly and dystrophin intracellularly, linking the matrix to the cytoskeleton. Mutations in dystrophin underlie Duchenne muscular dystrophy (DMD), and mutations in the transmembrane DGC components called sarcoglycans (a -, b -, g -, and d -) lead to autosomal recessive limb girdle muscular dystrophies (LGMD 2C-F). Thirdly, laminin 2 is a principal ligand for the major integrin of muscle, a 7b 1, loss of which also

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B.L. Patton et al. / Neuromuscular Disorders 9 (1999) 423±433

Table 1 Laminin chains in normal and dystrophic human muscle a Dx

n

Age at biopsy (years)

a2

1

Normal

9

0.02±023

1 1 1

2 3

CMD-a 2

3 1

0.3, 8, 17 0.6

# #

4 5 6

CMD 1 a 2

2 3 2

2, 4 3, 5, 11 14, 16

7 8 9 10 11

DMD

1 6 4 1 1

12

LGMD-2D

13 14 15 16 17 18

LGMD

19 20

EDMD

c

b

a4

a5

b1

b2

g1

a 1, a 3, b 3, g 2

±

±

1 1 1

±,^

1 1 1

±

Note

" "

ˆ "

ˆ ˆ

ˆ ˆ

ˆ ˆ

ˆ ˆ

A, B, C d D, E

ˆ ˆ ˆ

ˆ ˆ ˆ

" ˆ ˆ

ˆ ˆ #

ˆ ˆ ˆ

# ˆ ˆ

ˆ ˆ ˆ

E E E

1.3 2±7 2±7 7 9

# ˆ ˆ ˆ ˆ

ˆ ˆ ˆ (") ˆ

" " " " ˆ

# ˆ ˆ ˆ ˆ

ˆ ˆ " ˆ "

# ˆ ˆ ˆ ˆ

ˆ ˆ ˆ ˆ ˆ

F

1

9

ˆ

ˆ

"

ˆ

"

ˆ

ˆ

H

2 3 2 4 1 2

3, 5 6, 8, 58 6, 14 22±60 40 20, 23

ˆ ˆ ˆ ˆ ˆ ˆ

ˆ ˆ ˆ ˆ ˆ ˆ

ˆ ˆ " " ˆ ˆ

# ˆ ˆ ˆ # #

ˆ " " ˆ ˆ "

# ˆ ˆ ˆ ˆ ˆ

ˆ ˆ ˆ ˆ ˆ ˆ

I J

2 1

6, 41 14

ˆ ˆ

ˆ ˆ

ˆ "

ˆ #

" ˆ

ˆ ˆ

ˆ ˆ

G

K

a

Dx, clinical diagnosis; a 1±5, b 1±3, g 1±2, immunoreactivity for respective laminin chain(s). 1 1 1 , strong immunostaining; ^ , very weak staining; ±, none detected; ˆ , equivalent to normal. c # , " , decrease, increase in intensity of staining compared to normal controls. d A, complete absence of a 2 observed with two independent antibodies; B, 17-year biopsy from patient described in [27]; C, 8-year biopsy from patient described in [19]; D, complete absence of a 2 observed with one antibody; decrease in a 2 observed with other antibody; E, patient described in [27]; F, marked variability among ®bers; G, a 4 levels were lower than observed in CMD patients; H, patient described in [27,44]; I, siblings; J, 6-year biopsy from affected son of patient in line 18; K includes biopsy from mother of patients in line 13. b

leads to dystrophy in humans and mice [10,11]. Fourthly, and most important, mutations in the laminin a 2 gene underlie approximately 50% of the cases of congenital muscular dystrophy (CMD), as well as the dystrophy observed in dy/dy mice [12±20]. Although initial studies of laminin in muscle used antisera that recognized multiple subunits, several groups have now examined the distribution of individual laminin chains in dystrophic muscle of various types. The most striking result of these studies is the loss of laminin a 2 in CMD, but alterations in the apparent abundance of other chains have also been reported [21±33]. Nonetheless, several questions remain unanswered. Firstly, dystrophic muscle has not yet been tested for expression of the recently discovered a 3, a 4, b 3 and g 2 genes [34±38]. Of these, the a 4 chain is of particular interest, in that it is expressed by embryonic muscle and upregulated in adult a2-de®cient mice [39]. Secondly, the a 5 chain is upregulated in diverse dystrophies [21±25,27±29,32,33], but it is not clear whether this re¯ects a response to myopathy or is a consequence of immaturity, denervation or regeneration [21,25]. Thirdly, levels of b 1 chain are decreased in muscles of some dystrophic patients

[21±24,28±31], but it remains uncertain whether levels of b 1 discriminate among dystrophic subtypes. Fourthly, both decreases and increases in levels of b 2 have been reported to accompany Walker±Warburg and congenital muscular dystrophies [26,29,33], but it remains unclear whether these changes are speci®c to these dystrophies or are also present sporadically in other patient populations. Here, as a ®rst step in addressing these questions, we used subunitspeci®c antibodies to assess the distribution of all ten known laminin chains in muscles from dystrophic humans and from embryonic, adult, denervated, regenerating and dystrophic mice.

2. Patients, materials, and methods 2.1. Patients Patients are listed in Table 1. A diagnosis of CMD was assigned to 11 children who had diffuse muscular weakness in the ®rst year of life, developed contractures by 2 years of age, and had muscle biopsies with diffuse endomysial ®bro-

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sis and variation in muscle ®ber size. This group was divided into children with laminin a 2-de®ciency (n ˆ 4) and those with normal levels of laminin a 2 (n ˆ 7). The children with a 2-de®ciency had more severe weakness and none achieved independent ambulation. In contrast, all children with preserved laminin a 2 achieved independent ambulation. Emery±Dreifuss muscular dystrophy (EDMD) is a linked recessively inherited disease characterized by progressive humeroperoneal weakness, contractures at elbows, wrists, ankles, and neck extensor muscles, and cardiac arrhythmias in the second and third decade of life [40]. Three male patients presented with characteristic weakness and contractures and were immunohistochemically negative for emerin, the product of the gene mutated in EDMD [41,42]. A diagnosis of LGMD was assigned to 15 patients who showed onset of slowly progressive, proximal greater than distal, muscle weakness after the age of 2 years; and had dystrophic muscle biopsies that stained normally for dystrophin. Contractures were variable. One female patient with a clinical diagnosis of LGMD was shown to have primary asarcoglycan de®ciency [19,43,44]; at biopsy, she had proximal-greater-than-distal muscular weakness but was ambulatory. Other LGMD patients studied showed normal a sarcoglycan staining and therefore did not have LGMD 2D. In two families with LGMD, a history indicative of dominant inheritance was present. One female patient had two affected daughters (ages at biopsy: 2.8 and 4.8). One male patient had an affected son. Thirteen boys with DMD presented before age seven and were dystrophin-de®cient on biopsy. Nine normal biopsies obtained from patients aged 0.02± 23 years lacking obvious neuromuscular disorders were studied as controls. 2.2. Animals Mdx (dystrophin-de®cient), dy/dy (C57BL/6J-Lama2 dy) and wild-type C57BL6 mice were obtained from Jackson Laboratories (Bar Harbor, ME). Utrophin 2/2 mutant mice were generated and bred to mdx mice in our laboratory [45], and generously provided for this study by Dr R.M. Grady. Muscle denervation and damage were separately performed using anesthetized 4-week-old mice. The left sternomastoid muscle was denervated by resecting a portion of the nerve proximal to the neighboring cleidotrapezius muscle. To induce muscle regeneration, the sternomastoid was damaged by repeated crushes with forceps. Mice were sacri®ced 16 days following denervation, and 5 or 10 days following muscle crush. Operated and contralateral control muscles were dissected and pinned at full extension in OCT medium, with control muscles above and in register with operated muscles. Frozen blocks were marked to indicate the locations of muscle origin, insertion, and endplate

425

regions, then trimmed to expose the extrasynaptic regions of the muscle for cross-sectioning. 2.3. Histochemistry Muscle biopsies were ¯ash-frozen in liquid nitrogencooled isopentane, and stored at 2 708C prior to cryostat sectioning. Serial sections were stained with hematoxylin and eosin, modi®ed Gomori's trichrome, esterase, Sudan black, and myosin adenosine triphosphatase (ATPase) at pH 9.4, 4.6, and 4.3 [46,47]. Diagnostic immunohistochemistry was performed with mouse monoclonal antibodies to the mid-rod, carboxyl-terminus, and amino-terminal epitopes of dystrophin [48], and to a -sarcoglycan (Vector Laboratories; Burlingame, CA), and with a polyclonal antiserum speci®c for emerin (residues 168±178; A. Pestronk, unpublished). The following antibodies to laminin were used to stain human muscle: A rabbit antiserum was raised to a bacterially-produced fusion protein containing amino acids 646± 911 of human laminin a 1 (GenBank Accession #X58531; [49]). The laminin a 1 cDNA fragment encoding those amino acids was obtained by reverse transcription-coupled polymerase chain reaction and cloned into an expression vector (pET-23a; Novagen, Madison, WI), using methods described previously [2]. Rabbit antiserum to recombinant G-domain of human a 2 [50] was kindly provided by Dr Peter Yurchenco (Robert Wood Johnson Medical School, Piscataway, NJ). Monoclonal antibody BM165 to human a 3 and a polyclonal antiserum to laminin-5 (which recognized the a 3, b 3, and g 2 chains) were kind gifts of Drs Robert Burgeson (Massachusetts General Hospital, Boston, MA) and M. Peter Marinkovich (Stanford University, Palo Alto, CA). Rabbit antiserum to recombinant a 4, mouse monoclonal antibodies C1 and C4 to b 2, and mouse monoclonal antibody D18 to g 1 were generated in our laboratory and described previously [2,5,51]. Monoclonal antibodies 5H2 to a 2, 4C7 to a 5 and 4E10 to b 1 were generous gifts of Dr Eva Engvall (Burnham Institute, La Jolla, CA; see [4]), and are also available commercially from Gibco/ Life Technologies (Gaithersburg, MD). The 4C7 antibody was originally thought to recognize the a 1 chain [4,5] but has subsequently been shown to recognize a 5 instead [32,52]. We have reinterpreted published data as warranted [21±25,28,29,33] based on this reassignment. The following antibodies to laminin were used to stain mouse muscle: Rat monoclonal antibody 198 to mouse a 1 was a kind gift of Dr Lydia Sorokin (Erlangen, Germany; [53]). Rabbit antisera to a 2, a 4, and kalinin were as described above. Rabbit antiserum to a 5, and guinea pig antisera to b 2 were generated in our laboratory [2,5]. Rat monoclonal antibody 5A2 was obtained from Dr Dale Abrahamson (University of Alabama, Birmingham, AL; [54]), and shown by us to recognize b 1 [55]. Rat monoclonal

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B.L. Patton et al. / Neuromuscular Disorders 9 (1999) 423±433

Fig. 1. Laminin chains of normal human muscle (a±j). Serial sections from 2-year-old human muscle were stained with antibodies speci®c for the indicated laminin chains. Muscle ®ber BL is rich in a 2, b 1, and g 1. The a 4, a 5, and b 2 chains are not detectable in muscle BL, but are associated with some blood vessels, including capillaries (arrows), venules and arterioles (arrowheads). (k) Section from 23-year-old human muscle stained with antibody speci®c for the ubiquitous laminin g 1 chain. Laminin-containing BLs are associated with endoneuria (en), perineuria (pn), arterioles (a), venules (v), capillaries (c), myo®ber surfaces (m), neuromuscular junctions (nmj), and Schwann cells (s) which cap the nerve terminal. Kal, antibody to laminin-5 a 3b 3g 2, originally kalinin), which recognizes epitopes on a 3, b 3 and g 2 chains. Ctl, no primary antibody. Bar in j is 30 mm for a-j and 45 mm for k.

antibody 1914 to mouse g 1 was purchased from Chemicon (Temecula, CA). Human and mouse muscle sections were stained and photographed as previously described [39]. Brie¯y, sections were incubated with primary antibodies at appropriate dilutions in PBS containing 20 mg/ml BSA and 2% normal goat serum for 4 h at RT, or overnight at 48C, and unbound antibodies removed by repeated washing in PBS over 30 min. Bound monoclonal antibodies were detected using ¯uorescein-conjugated goat anti-rat or -mouse IgG (BMB). Rabbit polyclonal antibodies were detected with Cy3-conjugated goat anti-rabbit Ig (Cappell-Organon, Tekni, VA). Following a 1-h incubation, unbound second antibodies were washed away in PBS, and the stained sections mounted in glycerol containing p-phenylenedrimine (1 mg/ml) to retard photo-bleaching, and stored at 2208C. Staining was assessed within 48 h. Images were acquired using a cooled-CCD camera (Photometrics, Princeton, NJ) using low light levels which did not appreciably photo-bleach the samples. For each primary antibody, expo-

sure times were held constant throughout the sample series. Control and dystrophic muscles were stained and photographed in parallel. Findings were con®rmed through additional rounds of staining. 3. Results 3.1. Normal human muscle Biopsies of normal muscle were obtained from nine individuals, ranging in age from 1 week to 23 years. Cryostat sections were stained with antibodies speci®c for the ten known laminin chains (Fig. 1). The BL surrounding all skeletal muscle ®bers was strongly stained by antibodies to the a 2, b 1, and g 1 chains. Antibodies to a 1, a 3, a 4, a 5, b 3, and g 2 did not detectably stain muscle ®ber surfaces. Anti-a 4 and anti-a 5 did stain other intramuscular structures, including blood vessels (arrows and arrowheads in Fig. 1). No a 1-, a 3-, b 3- or g 2-like immunoreactivity was detectable in muscle but the activity of these antibodies was con®rmed by showing that they stained structures in

B.L. Patton et al. / Neuromuscular Disorders 9 (1999) 423±433

427

Fig. 2. Laminin a chains in dystrophic muscle. (a±e) 2-year-old DMD; (f±j) 0.6-year-old a 2 (merosin)-de®cient CMD. (k±o) 11-year-old a 2-positive CMD. Laminin a 2 is undetectable in a 2-de®cient CMD but unaffected in DMD and a 2-positive CMD. Laminin a 4 is present in blood vessels in all samples, but speci®cally upregulated in myo®ber BL in a 2-de®cient CMD. Laminin a 5 is present in BLs of some muscle ®bers in a variety of dystrophies. Neither laminin a 1 (left panels) nor a 3 (not shown) is detected in dystrophic muscles. Right panels show staining with anti-g 1, which marks all BLs. Some stained capillaries and small blood vessels are indicated (arrows). Bar in o is 40 mm.

sections from human kidney or lung (data not shown; [2]). Anti-b 2 did not detectably stain muscle ®bers from the youngest patients (,1 year of age), but strongly stained blood vessels or perineurium in all samples and lightly stained some muscle ®bers in older patients. These results are consistent with the conclusion [4,5,8] that laminin 2 (a 2/b 1/g 1) is the major laminin of muscle ®bers throughout postnatal life. 3.2. Dystrophic human muscle Biopsies of dystrophic muscle were obtained from 42 patients. Based on criteria detailed above, this series included individuals with CMD, DMD, LGMD, and EDMD. As with normal muscles, cryostat sections were stained with antibodies speci®c for all laminin chains known to date (Table 1, and Figs. 2and 3 ). All dystrophic muscles resembled controls in that their BLs lacked laminins 1, a 3, b 3, and g 2. However, levels of the other six laminin chains differed signi®cantly between controls and at least some dystrophic muscles. The differences were as follows: Firstly, levels of laminin a 2 were completely undetectable in three CMD patients and markedly reduced in a fourth patient. The loss was speci®c in that levels of a 2 were normal or nearly normal in all other dystrophic patients. In three of the CMD patients, a 2 was undetectable both with a monoclonal antibody recognizing an epitope within the carboxyl-terminal 80 kDa fragment of a 2 and with a polyclonal antiserum that recognizes both the 80 kDa C-terminal and the N-terminal 300 kDa portions of the

molecule (P. Yurchenco, personal communication). In the fourth patient, the monoclonal antibody detected no antigen whereas the polyclonal antiserum showed clear immunoreactivity, but at reduced levels compared to normal controls. This fourth patient therefore lacks a C-terminal epitope but is not completely de®cient in a 2. Antibodies speci®c for the 300 kDa fragment were not available to test its presence, but our results are similar to those in several recent reports on partial deletion of the laminin a 2 gene in some a2-de®cient CMD patients [33,56,57]. Secondly, laminin a4 was present at markedly elevated levels in muscles of all a 2-de®cient CMD patients. This elevation was selective in that a 4 was not detectable in any normal muscle, in any of the LGMD, EDMD or a 2positive CMD patients, or in 12 of 13 DMD patients. Laminin a 4 was present in muscles of the 13th DMD patient, but at lower levels than in any a 2-de®cient CMD muscle. Thus, there is a high albeit not absolute, inverse correlation between levels of a 2 and a 4 in adult human muscles. Thirdly, laminin a 5 was readily detectable in muscles from 23 of 42 dystrophic patients. This elevation was nonselective in that at least one biopsy from each of the diagnostic categories of dystrophy examined (CMD, LGMD, DMD, and EMD) contained a 5-positive ®bers. A majority of myo®bers expressed a 5 in most of these biopsies, but levels varied among ®bers within each specimen. In contrast, no myo®bers were signi®cantly a 5-positive in the remaining dystrophic samples or in any of the normal muscles. Fourthly, levels of b 1 were markedly decreased in ten of the 42 dystrophic muscle biopsies. The six samples with the

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B.L. Patton et al. / Neuromuscular Disorders 9 (1999) 423±433

were b 2-positive. In contrast, no patients with CMD had levels of b 2 detectably increased compared to controls.

Fig. 3. Laminin b chains in dystrophic muscle. (a±c) 2-year-old DMD; (d± f) 11-year-old a 2-positive CMD; (g±i) 23-year-old LGMD; (j±l) 14-yearold EDMD; (m±o) 6-year-old EDMD. Laminin b 1 levels are markedly decreased in some patients, including the LGMD and 14-year-old EDMD patients shown here although it was present at normal myo®ber BL levels in small blood vessels and capillaries. Laminin b 2 is absent or weakly detectable in normal muscle BL, but it is upregulated in some patients, including the LGMD and 6-year-old EDMD patients shown here. Laminin b 2 is readily detectable in perineurium and small arteries regardless of levels in muscle BLs. Right panels show staining with anti-g 1, which marks all BLs. Arrows mark examples of staining in capillaries and venules; arrowheads mark small arteries (in b) and perineurium (in e). Bar in o is 40 mm.

most marked decreases in b 1 were from patients with a 2positive CMD or LGMD, as were two of the four samples with more modest increases. Thus, our results are consistent with and extend previous reports that b 1 is selectively reduced in a subset of CMD and LGMD patients [22± 24,28±31]. De®ciency of a -sarcoglycan (adhalin) in LGMD-2D is accompanied by speci®c decreases in laminin b 1 in most reported cases, with lower levels of b 1 appearing at more advanced stages of disease [22,24,28]. In our series, however, the single a -sarcoglycan-de®cient biopsy had normal levels of b 1. This patient also had elevated levels of laminin a 5, which has previously been reported in a single, clinically-asymptomatic case [28]. One of the remaining patients with reduced b 1 was categorized as DMD and the other as a 2-de®cient CMD. Previous studies have not remarked on decreases in laminin b 1 in these diseases. Finally, increased levels of b 2 were present in 15 of the 42 dystrophic biopsies. Consistent with a previous observation [24], this group included the patient with LGMD-2D. In addition, subsets of patients with DMD, LGMD, and EDMD

3.3. Embryonic, damaged, and denervated mouse muscle Changes in laminin chain composition in hereditary human dystrophies might result from speci®c genetic lesions, but could also re¯ect necrosis, regeneration, or denervation of muscle ®bers. We addressed these possibilities in mice, where muscle regeneration and denervation can be experimentally induced. As previously reported, adult mouse muscle contains only the a 2, b 1 and g 1 chains (Fig. 4h±n) [39]). Antibodies to a 2, a 4, and laminin-5 chains, which are cross-reactive with mouse and human antigens, stained muscles identically in both species. In embryonic muscle, b 1 and g 1 are the only detectable b and g chains of those we assayed, but the complement of a chains is enlarged: a 4 and a 5 are present throughout the basal lamina of embryonic myotubes in addition to a 2 (Fig. 4a±g; [39,58]). Therefore, expression of either a 4 or a 5 in dystrophic muscle could re¯ect immaturity. During regeneration of muscle, some genes expressed in embryonic myotubes are re-expressed [59]. We speculated that the LAMA4 or LAMA5 genes might be among them. To test this possibility, we mechanically damaged the sternomastoid muscle in 1-month-old mice. Following such damage, muscle ®bers die and are phagocytosed, then new myotubes regenerate from satellite cells [60]. Regeneration occurs rapidly, with some myotubes formed within 2 days of damage and many by 5 days after damage. We stained cross-sections of regenerating muscles for the embryonic isoform of myosin heavy chain to mark areas of active regeneration. Embryonic myosin heavy chain was present throughout the muscle 5 days after damage, but was greatly downregulated by 10 days (Fig. 4o,v). Thus, regenerating ®bers matured rapidly. During the early stage of regeneration (5 days after damage), most muscle ®ber BLs contained substantial levels of a 5, along with a 2, b 1 and g 1 (Fig. 4q,s,t). Little or no a 4 was detected in any myotube BL (Fig. 4), despite the abundance of this chain in embryonic muscle (see above). However, low levels of a 1 were present in a subset of myotube BLs. The a 1-positive cells were restricted to the central core of damaged muscle, which is delayed in regeneration [61,62]. This result is consistent with our previous studies of embryonic development [39], which showed that expression of a 1 is restricted to the earliest stages of myogenesis, prior to the stage shown in Figs. 4a±g. By 10 days after muscle damage, a 1 was completely undetectable and a 5 was weakly detectable (Fig. 4w±b(). Thus, in mice, a 5 is selectively re-expressed along with a 2 during periods of active regeneration and then downregulated as muscle ®bers mature. Regeneration is generally accompanied by transient denervation. To determine whether upregulation of a 1 and a 5 resulted from denervation rather than regeneration,

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429

Fig. 4. Laminin chains in normal, regenerating, and denervated mouse muscle. (a±g) Embryonic (E15) muscle. (h±n) Adult muscle. (o±u) Regenerating muscle, 5 days after crush. (v±b 0 ) Regenerated muscle, 10 days after crush. c 0 -i 0 ) Denervated muscle, 16 days after nerve section. Muscles were stained with antibodies speci®c for embryonic myosin or individual laminin chains as indicated. Images from embryonic, regenerating, and denervated muscles include identical ®elds-of-view from sections double-stained with anti-a 1 and a 2, or with anti-a 5 and b 1. Embryonic MHC was present in all muscle ®bers in embryos, and was transiently re-expressed in adult muscles following injury. Laminin a 2 and b 1 were highly expressed by muscle ®bers in all samples, and a 4 and a 5 were expressed in embryonic muscles. Levels of a 5 increased transiently in regenerating muscle ®ber but levels of a 4 and a 2 did not change signi®cantly with muscle regeneration. Denervation alone produced no signi®cant changes in laminin expression. Similar results were observed after 7 days denervation (not shown). Despite their absence from myo®ber BLs in some muscles, a 4, a 5, and b 2 were detected at high levels in perineurial and vascular BLs: in adults, capillaries and venules (small arrows) had a 4 and a 5, arterioles (arrowheads) had primarily a 5 and b 2, and perineurium (pn) contained a a 4, a 5, and b 2. In all muscles, g 1 was present at normal levels, while the a 3, b 3, and g 2 chains were absent (not shown). Bar in i is 15 mm in a-g, and 40 mm in h-i.

we tested muscles that were surgically denervated but not directly damaged. The complement of laminin chains in denervated muscle did not differ detectably from that in normal adults (Fig. 4c 0 ±i 0 ). 3.4. Mouse models of DMD and CMD We assayed the laminins of skeletal muscle in two genetically de®ned mouse models of muscular dystrophy. Dystrophin 2/2 utrophin 2/2 double mutants have recently been introduced as a model of DMD; these mice more accurately phenocopy symptoms of Duchenne dystrophy than the

dystrophin mutant mice (mdx), perhaps because utrophin can compensate for loss of dystrophin more effectively in mice than in humans [45,63]. Dy/dy mice, which bear a still unidenti®ed mutation in the Lama2 gene, are a model for laminin a 2-de®cient CMD [12,13,15]. Laminins have not previously been studied in the mdx:utrophin 2/2 double mutants; we reported previously on laminins in dy/dy muscle [39], and extend those observations here. In skeletal muscles of mice lacking dystrophin and utrophin, the BLs contained readily detectable a 5; other laminin chains were present at normal levels (Fig. 5a±g; cf. Fig. 4h±

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a 2, b 1, and g 1 which were present at nearly uniform levels throughout the muscle. This distribution is similar to that seen in most DMD muscles (see above). No abnormalities were detected in the extrasynaptic BL of utrophin 2/2 muscle, but increased levels of a 5-imunoreactivity were present in the extrasynaptic BL of mdx muscle (data not shown). In dy/dy skeletal muscle, levels of a 2 were decreased and those of a 4 were increased compared to controls (Fig. 5j,k). In some limited areas, immunoreactivity for a 1 was also seen (data not shown). The distribution of other chains did not differ signi®cantly from the normal pattern (Fig. 4h±n and Fig. 5). Speci®cally, levels of a5 were substantially lower in dy/dy muscle than in mdx:utrn 2/2 mutant muscles. Thus, mice de®cient in laminin a 2 show a compensatory change in laminin isoform production that is similar to that in a 2-de®cient humans, but distinct from mice and humans lacking utrophin and/or dystrophin.

Fig. 5. Laminin chains in muscles of dystrophic mice. (a±g) Muscle from leg of a 14-week-old mdx:utrn2/2 double mutant. (h±n) Muscle from leg of a 2-month-old dy/dy mutant. Muscles were stained with antibodies speci®c for embryonic myosin or individual laminin chains as indicated. Sections were double-stained with anti-a 1 and a 2, and with anti-a 5 and (1, and identical ®elds-of-view are shown in these cases. A signi®cant subset of myo®bers in mdx:utrn2/2 muscles expressed embryonic MHCs and were judged to be actively regenerating; these were few and of small caliber in dy/dy muscles indicating a reduced frequency and altered pattern of myo®ber regeneration in this mouse. In both mouse dystrophies, myo®bers were variable in diameter and had centrally located nuclei (not shown). In mdx:utrn2/2 muscles, levels of a 5 were signi®cantly increased, while all other chains were unchanged. In dy/dy muscles, levels of a 2 were signi®cantly reduced and levels of b 4 similarly increased, while b 1 appeared only slightly reduced, and a 1, a 5 and b 2 were normal (i.e. absent). The a 4, a 5, and b 2 chains were detected in capillaries (small arrows), arterioles (arrowheads in e,l), or perineuria (arrowheads in g,n), as described in Fig. 4, except that a 5 was only weakly expressed in capillaries in the 8-week-old dy/dy muscles. In both dystrophies, the g 1 chain was present at normal levels, and a 3, b 3, and g 2 chains were absent (not shown). Bar in n is 40 mm.

n). Most myo®bers had signi®cant amounts of a 5, but expression varied considerably among ®bers, in contrast to

4. Discussion Several previous studies have assessed the distribution or abundance of individual laminin chains in the BLs of dystrophic human muscle. Most notably, many patients with CMD show markedly decreased levels of a 2 [15], and in many of these, the primary defect is now known to be a mutation in the a 2 gene [16,18,19]. In addition, several groups have reported increased levels of a 1, a 5, or b 2 chains or decreased levels of b 1 or b 2 in dystrophic muscle [21±30,32,33]. Here, we have extended this analysis in several ways: (1) We provide the ®rst data on the occurrence of the a 1, a 3, a 4, b 3 and g 2 chains in normal human muscle; (2) We evaluate the distribution of all ten known laminin chains in patients with CMD, DMD, EMD and LGMD; (3) We use mouse muscle to ask whether some of the changes observed in dystrophic human muscles might re¯ect immaturity, regeneration or denervation; (4) We directly compare human muscle with two mouse models of dystrophy: mdx:utrn 2/2 mice, which provide a model of DMD, and dy/dy mice, which serve as a model of a 2-de®cient CMD. From these results, we draw conclusions about the regulation of laminin chains in dystrophic muscle. 4.1. Laminin a 5 Laminin a 5 is absent from extrasynaptic regions of normal adult human and mouse muscle, but is present in the BL of many dystrophic ®bers. We believe that the presence of laminin a 5 in the BL of dystrophic muscle re¯ects regeneration, as previously proposed by others [21,23,25], rather than a disease-speci®c process. This conclusion is based on the increased levels of a 5 observed in diverse, genetically distinct human dystrophies and in genotypically normal regenerating mouse muscle. In both mice and humans, a 5 is abundant in the BL of embryonic myotubes, then disappears perinatally, except at synaptic sites [5,39,58,64]. During regeneration, however, this chain reappears rapidly and transiently: a 5 was readily

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detectable 5 days after damage, and was signi®cantly downregulated after an additional 5 days. Levels of a 5 were similar among neighboring ®bers in regenerating muscles, but varied considerably among ®bers in dystrophic muscles. Likewise, high levels of embryonic myosins [59] were present in most ®bers of genotypically normal muscles 5 days following whole muscle crush, which damages all ®bers synchronously, whereas embryonic myosin-positive (regenerating) ®bers in dystrophic humans and mice are interspersed with mature and necrosing ®bers (Fig. 5a); [45,63,65±67]). Thus, restriction of laminin a 5 to regenerating or recently regenerated myotubes may help to explain its irregular appearance in human dystrophy [11,23,25]. Levels of a 5 also varied among patients within speci®c disease subtypes; a minority of DMD, and a majority of CMD, LGMD, and EDMD patients had no detectable increase in a 5 expression. Our observations are consistent with and extend previous reports, which emphasize signi®cant increases in a 5 expression in many but not all dystrophic samples [21±25,27±29,32,33]. In our series, levels of a 5 were usually lower in CMD muscles than in DMD muscles. Likewise, in mice, levels of a 5 were substantially lower in dy/dy muscle than in mdx:utrn 2/2 muscle. These results are consistent with the ®nding that CMD is relatively nonprogressive despite its early severity and that muscle regeneration is prominent and ongoing in mdx:utn 2/2 mutant mice but not in dy/dy mice [45,68]. However, two of seven CMD patients in our study had increased levels of a 5, as did some patients in previous studies [15,21,23,29]. Thus, the association is not an absolute one. Further analyses and larger sample sizes may be necessary to understand what factors in addition to regeneration affect levels of laminin a 5 in speci®c dystrophies. 4.2. Laminin a 4 Laminin a 4 is absent from extrasynaptic regions of normal adult human and mouse muscle, but appears in the BL of a 2 de®cient (CMD) muscle. We believe that the appearance of a 4 in CMD is a speci®c response to loss of a 2 rather than a consequence of dystrophy or regeneration. This conclusion is based on three sets of results: the absence of laminin a 4 from myo®ber BLs in 37 of 38 dystrophic muscles with near-normal levels of a 2; the upregulation of a 4 in dy/dy mice, which have a mutation at the Lama2 locus [14]; and the absence of a 4 from regenerating ®bers in genotypically normal and mdx:utrn 2/2 mouse muscles. Although levels of a 4 and a 2 appear to be closely and inversely correlated, there may be factors that regulate a 4 expression independently of a 2. This is indicated by the presence of both a 2 and a 4 in one DMD patient, and by the observation that a 2 and a 4 are co-expressed in embryonic mouse muscles [39]. It is also noteworthy that a 4 and a 5 are both expressed in developing muscle but only the latter reappears during regeneration. Thus, whereas the appearance of a 5 is associated with active muscle ®ber

431

regeneration, levels of a 4 may be independently regulated in adults by an a 2-dependent signal. 4.3. Laminin b 1 and b 2 Many dystrophic biopsies showed decreased staining with anti-b 1 and/or increased staining with anti-b 2 compared to controls. We believe that these changes are secondary to myopathy and are therefore not yet useful either diagnostically or etiologically. We base this conclusion on three sets of results: the sporadic occurrence of altered b 1 and b 2 levels in subsets of patients with diverse dystrophies; the absence of such changes from genetically de®ned mouse models (dy/dy and utrn:mdx); and the lack of correlation with physiologically de®ned states of immaturity, denervation or regeneration. Decreases in b 1 were observed in ®ve of 14 LGMD patients and in two of seven a 2-positive CMD patients. These results are consistent with previous reports of decreased b 1 levels in subsets of LGMD and CMD patients [22±24,28,30,31,69]. In addition, b 1 levels were decreased in one of three EDMD patients. In contrast, patients with DMD and a 2-negative CMD did not frequently show decreased b 1 (1 of 13, and 0 of 4, respectively). Thus, decreased b 1 levels are neither generally present in the patient population nor restricted to a single category. Nonetheless, there is a clear preferential association with some dystrophic types. Increases in b 2 were observed in ®ve of 13 DMD patients, eight of 15 LGMD patients and two of three EDMD patients, but in none of the 11 CMD patients. Increases in b 2 have previously been reported in some but not all cases of LGMD [24]. Increased b 2 sometimes accompanied decreased b 1, but this association was infrequent (seen in only two cases). Thus, our results do not support the simple hypothesis that levels of b 2 increase to compensate for loss of b 1. Cohn et al. have recently reported that b 2-like immunoreactivity is present in extrasynaptic BL of normal muscle, and decreased in muscles of some dystrophic patients, including those with b 2-de®cient CMD and Walker± Warburg syndrome [26,29,33]. Reports on Walker± Warburg syndrome, however, include one report of decreased b 2 [26] and a second in which no decrease was observed [33]. In our series, b 2-like immunoreactivity was barely detectable in normal myo®ber BL, although reactivity was intense in arterial, perineurial, and synaptic BLs. Thus, although some b 2 may be present in extrasynaptic BL, its levels are extremely low compared to those at synaptic sites and in other BLs. This pattern is similar to that documented previously by us [5] and others [24,70]. Several factors may contribute to this discrepancy, including our insensitivity to low expression levels, variation in b 2 expression among populations, and the fact that a commonly-used anti b 2 monoclonal antibody, C4, crossreacts to a minor degree with b 1 [5,71]. Lack of signi®cant b 2-like immunoreactivity in normal muscle prevented us

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from scoring decreases in dystrophic muscle. In any event, our results emphasize the large variability in b 1 and b 2 levels in myo®ber BLs seen in among dystrophic populations.

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