Does utrophin expression in muscles of mdx mice during postnatal development functionally compensate for dystrophin deficiency?

162

.hmnud o! the Neurologwal S(';em, ~. 122 ~lt~t~) 1~2 [ 7!~ ~7 1994 Elsevier Science B.V. All rights resel-ved1t~22-5II)X. t~4./$(~7.(1t~

JNS 4254

Does utrophin expression in muscles of mdx mice during postnatal development functionally compensate for dystrophin deficiency? F. P o n s

a,b,* A .

Robert

a, J . F . M a r i n i c a n d J . J . L 6 g e r ~

"Pathologie Moldculaire du Muscle, INSERM U300, Facult~ de Pharmacie, Ar'enue Ch. Flahaut, F-34060 Montpellier Cedex I, France. b Pathologic G~n&ale, Facultd de M~decine, Place Henri IV, F-34000 Montpellier, France, c Unitersit~ Aix-Marseille H / UFRSTAPS & CNRS UPR 418, Unitd de Neurocyberndtique Cellulaire, 280 Bd. Ste. Marguerite. F-13009 Marseille, France

(Received 26 May, 1993) (Revised, received 28 September, 1993) (Accepted 30 October. 1993) Key words: Utrophin; mdx muscle; Slow and fast muscles; Development

Summary We correlated utrophin expression with the physiopathological course in mdx mice. Evolution of the pathology was assessed by monitoring expression of developmental MHC in mdx mice versus control. Utrophin expression is detected by dystrophin/utrophin cross-reacting antibodies and can only be evaluated in mdx mouse muscles (in absence of dystrophin). This protein was expressed at the periphery of all myotubes and myofibers during the first postnatal week. It began declining in fast muscles before the third week and disappeared from the soleus between the 3rd and the 4th week. The decrease was concomitant with a sudden degenerative/regenerative process affecting slow muscle earlier and more massively than fast muscles. The pathological process became stable in all muscle types (except the diaphragm), with greater utrophin expression in the soteus. These results in mdx mice along with observed utrophin expression in severely affected DMD patients suggest that overexpression of utrophin is not enough to explain the stability of regenerated fibers in mdx mice.

Introduction Unlike D M D patients, who die during the second decade of their lives following a dramatic course, mdx mice with the same genetic defect and the same absence of dystrophin are surprisingly able to overcome the disease. The mdx mouse has no obvious disability and the same longevity as unaffected controls (Dangain and Vrbov~i 1984; Carnwath and Shotton 1987; Coulton et al. 1988; Cullen and Jaros 1988; Cutlen and Walsh 1988). However, in both mdx mice and D M D patients the absence of dystrophin, which is the primary cause of the pathology, induces sequences of events that include reduction or disappearance of several components of the dystrophin/glycoprotein complex (Ervasti et al. 1990), and fiber necrosis. Progressive impairment of muscles in D M D patients is not

* Corresponding author. Tel.: 67 04 16 65; Fax: 67 04 21 40. Abbreuiations: DRP: dystrophin related protein or dystrophin

homologue; MHC: myosin heavy chain; DMD: Duchenne muscular dystrophy; ELISA: enzyme-linked immunologicsolid assay; ATPase: adenosine triphosphate hydrolase; IgG: immunoglobulin G; EDL: extensor digitorus longus. SDS-PAGE: sodium dodecyl sulfate-polyacrylamide gel electrophoresis; kDa: kilodalton. SSDI 0022-5t0X(93)E0265-B

efficiently compensated by regeneration of the muscle fibers, whereas regeneration is successful in mice. This suggests that newly regenerated fibers could be more stable and the functional properties of these muscles appear to be only slightly impaired (Coulton et al. 1988; Cullen and Walsh 1988; Dimario et al. 1991). As shown in several studies, the inability of dystrophin-deficient human muscles to successfully regenerate cannot be explained by a lack or exhaustion of satellite cells (Wakayama et al. 1979; Blau et al. 1983: Terassawa 1986; Watking and Cullen 1986). Two observations have led to new hypotheses. The first observation concerns detection of a homologous dystrophin transcript encoded by chromosome 6 in fetal muscle (Love et al. 1989). The homology to dystrophin originally found in the C terminal part has now been extended to the entire molecule (Tinsley et al. 1993). The corresponding protein or dystrophin related protein ( D R P ) was recently designated as "utrophin'" (Khurana et al. 1990; Heliwell et at. 1992). The protein was first immunolocalized in dystrophin deficient models at the N M J (Miike 1989; Fardeau et al. 1990; Pons et al. 1991; Ohlendrick et al. 1991). in vessels (Augier et al. 1992) and at the periphery o f muscle fibers (Voit et al. 1991; Man et al. 1991; HeUiwell et al. 1992:

Karpati et al. 1993). In addition, Koga et al. (1993) showed developmental expression of utrophin in norreal mice preceding the expression of dystrophin. In muscles wllere dystrophin is absent, up-regulation or re-expression of utrophin at the fiber periphery, has led many researchers to suggest that this protein may compensate for dystrophin deficiency (Takcmitsu c t a l . 1991: Khurana c t a l . 1991: Karpati et al. 1993). The second obsc~'ation (Dimario et al. 1991), based on the expression of developmental M H C as a reporter of the d e g e n e r a t i v e / r e g e n e r a t i v e process, noted the number o[ regenerating fibers decreased with age in mdx mice. These authors also suggested that the newly regcncralcd fibers are stable because of the balanced expression of utrophin. In the present study, we attcrnpt to go fttrther test this hypothesis by examining the expression of utrophin during maturation of a slow Isoleus) and various fast mdx mouse muscles. The concomitant development of the d e g e n e r a t i v e / r e g e n erative process was monitored by assessing the size and number of fibers expressing developmental MHC. The utrophin content was evaluated in three muscles: E D L and solcus which are compared in the present study ',tnd the diaphragm which is particularly affected in older mdx mice (Stedman et al. 1991; DupontVcrstecgdcn et al. 1902).

Materials and methods

.tnti-myosin heauy chain (MHC) monoclonal antibodies" Monoclonal antibodies were prepared from mice immunized with SDS-treated myosins of different hu-

man, bovine+ and rabbit skeletal and cardiac muscles. Three anti-MHC monoclonal antibodies were selectcd on the basis of their different reactivities toward adult and developmental M H C Anti-developmental M H C antibody (F158 4C10) was prepared with skeletal muscle M H C from a 17-week-old bovine fetus as antigen. Anti-slow M H C antibody (F36 4H3) was prepared with M H C from human ventricle. Anti-fast lla ( F l l 3 151:4) antibody was prepared with rabbit fast myosin as antigen. These monoclonal antibodies have been characterized m previous studies on humans or rats (Deschene et al. 1985; Marini et al. 1989). Their specificitics werc confirmed by comparing their rcactivitics in mice muscles of different ages. Threc approaches were used: first, immunofluorcscence labeling on ct~.,ost;.tt sections was comparcd to fiber classification in ATPascs on serial sections: then reactivities of thc diffcrcnt antibodies were analyzed by ELISA on total muscle extracts from mice of different ages: finally low porosity P A G E was used to separate the different MH('s ',rod the reactivity of different antibodies was tested by Western blot analysis. The specificities of these antibodies were almost identical in mice and other species.

Anti-dystrophin monoclonal antibodies Three anti-dystrophin monoc[onal antibodies were obtained by hybridization of lymphocytcs from two mice immunized with dystrophin fragments spanning two regions of chicken skeletal muscle dystrophin. Antibody H.5A3 was obtained using the entire dystrophin carboxy-terminal domain (residue 3357-3660), while two others, C.4GI0 and C.5G5, were obtained using a fragment of the dystrophin spectrin-likc rod-shapcd

Fig. 1, Serial frozen sections of normal soleus and fast adjacent muscles of an adult BLI0 mouse stained with either anti-dvstrophm (('.5(]5) (A) and u t r o p l m l / d y s t r o p h i n reacting antibodies ( ( ' 4 G 1 0 ) ( B ) both dircctly coupled to fluoresceinc: "'s'" refers to the soleus, and "'f'" 1o the adjacent fast muscle. Bofll :mtibodies stained the periphery of slow and fast fibers: the u t r o p h i n / d y s t r o p h i n reacting antibodies reacted more inlcnscly at the periphery of soleus fibers and also labeled blood vessels as shown by the arrow. Bar :: 20 ~tm.

164

domain (residues 1173-1728). These antibodies were selected using ELISA with their own antigens. They stained the periphery of normal human skeletal muscles, as well as mouse muscles (Fig. 1). In Western blot analysis, the antibodies detected only a 400 kDa band in normal human and mouse muscle extracts. Antibody C.5G5 only reacted with normal muscle and not with DMD or mdx mouse muscles. It is hereafter termed "anti-dystrophin antibody". In contrast, in mice or humans with a deletion at the Xp21 locus, antibodies C.4G10 and H.5A3 detected a single 400 kDa protein in muscle extracts, localized by histoimmunology at the neuromuscular junction (NMJ), at the periphery of the muscle fibers, and at the vascular level (Fardeau et al.

1990; Pons et al. 1991; Voit et al. 1991: Augier et al. 1992). Moreover, with ELISA, antibody H.5A3 reacted with the recombinant bacterial protein issued from the chromosome 6 encoded eDNA, identified as DRP (utrophin) by Khurana et al. (1990). The two latter antibodies were considered to be anti-dystrophin and they cross-reacted with the dystrophin homologue not derived from chromosome X. These antibodies are further designated as "utrophin/dystrophin reacting antibodies". Since all anti-mouse IgG used to detect monoclonal antibodies cross-reacted on mouse tissue, particularly at the periphery of skeletal muscle fibers, we labeled the three anti-dystrophin antibodies with fluorescein.

......................

---J

Fig. 2. Frozen sections of parts of soleus and adjacent (neighboring) fast muscles from 2 days (A), 2 weeks (B). 3.5 weeks (C). and adult t DI from mdx mice directly stained with utrophin/dystrophin reacting antibodies. The "s" refers to the soleus, and "f" to the adjacent fast muscle. Utrophin expression varied in intensity with age. Utrophin was present in all muscles at 2 days, was undetectable in fast muscles at 2 weeks and in both types of muscles at 3.5 weeks. Its reexpression was clear only in slow adult mouse muscles. Bar = 20/,tin.

Utrophin/dystrophin

reacting antibodies detect thc

T h e s e a n t i b o d i e s w e r e p r e v i o u s l y t e s t e d a c c o r d i n g to t h e m e t h o d o f B u r k o t et al. ( 1 9 8 5 ) f o r t h e i r a b i l i t y to

p r e s e n c e o f u t r o p h i n in mdx m o u s e m u s c l e s w h c r e n o

be coupled.

dystrophin

is e x p r e s s e d ( T a n a k a

et al. 199()). T h e re-

I:ig. 3. Serial frozen sections of whole soleus and parts of adjacent (neighboring) fast muscles from 2-week-old normal (A.(',E) and mdv~ mice (l:k D, F) slaincd \~ittl anti-slow (A,B). ~mti-fast lla (C,D) and anti-developmental MH(" (K,F). "'s'" refers to the soleus, and "'f'" to lhc adjaccnl fast muscle. The clistribulion of Mtt('s was identical in normal and in m~Z~ micc. Developmental MHC was still expressed ill many solcus fibers. [:lar 40/,zm.

l66

h

suits on serial sections of normal adult slow and fast mouse muscles with the two types of antibodies arc presented in Fig. i. Labelling was nearly the same with the two antibodies except that the utrophin cross-reacting antibody also stained vessels and fibers of the soleus more intensely. The hypothetical cross-reactivity of these antibodies, which are monoclonal, with another protein unrelated to dystrophin can be excluded for two reasons. First, no reactivity was observed in mouse muscles with dystrophin specific antibody labeled in the same conditions. Second, the described reaction was observed with two antibodies specific to two regions that structurally differ from the dystrophin molecule, one is specific to the C-terminal part of dystrophin which is structurally different from any known protein and the second is specific to the spectrin-like domain in the central part of the molecule.

Experimental animals mdx mice ( C 5 7 B L I 0 / S c S n mdx) and normal agematched control mice (C57BL/10ScSn)were used, The ages studied were 1, 2, 7, 9, 16, 21, 25, 28 days and adult (150 days). For the evaluation of utrophin in different muscles, three mice older than 1 year were used.

Immunofluorescence and tissue samples Muscles (4-6 muscle samples for each age) were dissected and immediately frozen in isopentane cooled to - 1 6 0 ° C in liquid nitrogen and stored at -80°C. The dissected hind limb muscles included the soleus, gastrocnemius, extensor digitorum longis (EDL) and plantaris. This dissection enabled us to compare slow and fast muscles in identical conditions. MHC detection was performed as described in Bouvagnet et al. (1985) by indirect immunofluorescence on 10-/zm serial transverse sections of different muscles~ Dystrophin and utrophin detection were carried out in a one-step reaction with fluorescein-labeled monoclonai antibodies.

Fiber counting The percentage of fibers containing developmental M H C was evaluated on several sections of soleus, plantaris, gastrocnemius and E D L muscles at different levels. A positive and even moderate fluorescent reaction was considered as being indicative of MHC expression.

Fig. 4. Serial frozen sections of parts of soleus and adjacent (neighboring) fast muscles from a 3.5-week-old mdx mice stained with anti-developmental (A), anti-slow (B) and anti-fast lla MHC (C). "s" refers to the soleus, and "f" to the adjacent fast muscle. Indicating a degenerative/regenerative progress, small myotubes expressing either developmental and fast lla MHC were present mostly in the soleus while none were present in fast muscles. Bar = 40 Izm.

I h7

Quantitative a/mlv.s'is o1" zm'ophitt content "Fhc relative amount of utrophin content was evaluated as described by Koga c t a l . (1992) for dystrophin evaluation, on diaphragm, solcus and ED1 muscles el 12-14-month-old mdx mice. The optical density of the immunorcactive utrophin band rcvcalcd by the antidystrophin/utrophin antibody ( C . 5 A 3 ) w a s measured in a Shimazu gel scanner. The amount of muscle tissue per extract was normalizcd referring to their myosin conlcnt c~aluatcd by densitomctry of the lower part of lhc gel stained with Coomtissic biilliant blue.

Results

Wc attempted to correlsltcd utrophin expression in slow and fast muscles with the pathological course in mdx mouse muscles. Wc invcstigated titrophin expression by immunohistochcmical analysis using monochmal utrophin /dystrophin reacting antibodies. In md_t mice, thcse antibodies detected utrophin in t h e a b s e n c e of dystrophin. The level of utrophin varied with age. It wcis initially cxprcsscd along the pcriphcry of all fibcrs during thc first wcck (Fig. 2A) and decrcascd lirst from the fast muscles (Fig. 2B). During this pc-tied. norma[ cind mdx muscles presented the same changes: suppressing dcvclopmcntal MHC expression. first in ttlC fast muscle and then in thc slow muscle. This period ended with thc same mosaic M H ( ' distriImtion pattcrn m 2-wcck-old normal and #mix muscles. ,is shown in t:ig. 3. The delayed maturity of the solcus compared to fast muscles was shown by the high ntmlbcr of dc\clopmcntal MHC containing fibers, already absent in fast illusclcs. Solcus mouse musclcs, as ellready dcmonstratcd in rat. only contain slow and fast lla MHC (Narusawa ct al. 1987) and developmental MH(" is qill prcscnt :,it lhis stage. Wc found that

t

#

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i

adjacent fast muscles contained list (Fig. 3(', D) and llb MtIC (fibers that did not react with any of the antibodies presented), and a fc~v slo~ fibers sc
,b J

# A

" II~

li~, i

,,i I'%

.

.

.

.

. ~

* .-i i

I:ig. 5. Sclia] fr~lcn scclioI}st)l aduh gastrocncmuis md.~. mou,,c i'tlttsclc_'sslaincd cithcl ,,,,it[i utrophm, d)
4(] ~L~Ill

I ¢)8 100 ~

cgJ

=

m

control soleus

~.EE

80

==~:I ,~x,~E

control f a s t

~1~

~IE 4o

o 1

2

3

4

8

20

Age in weeks

Fig. 6. Percentage of fibers containing developmental M H C (Dev.) in normal and in mdx slow and fast muscles. The number of fibers reacting with anti-developmental M H C antibody was determined by counting immunoreactive and total fibers in muscle sections at the indicated ages. T h e results for fast muscles were pooled from three muscles (gastrocnemius, plantaris and EDL). Values represent average percentages from two or three animals in which 200-400 fibers were counted. As the three fast muscles studied exhibited similar responses to the pathology, we often refer to fast muscles (gastrocnemius, plantaris, EDL) versus soleus.

lization of the degenerative/regenerative process. Only a few small loci of regenerating fibers reacting with developmental MHC (in groups of 2-20 fibers) persisted in all muscles (Fig. 5A), and only a few of them expressed utrophin. According to Dangain and Vrbovfi (1984) this process is achieved and the pathology is stabilized after 7 weeks. The MHC distribution is slightly altered in mdx muscles (Carnwath and Shotton 1987). The estimated proportions of fibers expressing developmental M H C (Fig. 6) indicated three periods in the course of the pathology: in the first period (1-2 weeks) before any degenerative process occurred, normal and mdx mouse muscles expressed and suppressed developmental MHC, first in slow muscle then in fast ones. In the second period (3-8 weeks) the pathology affected the slow and fast muscles differently. The peak of this process occurred at 3 weeks for the soleus and at 4 weeks for fast muscles. In summary, the degenerative/regenerative process affected slow muscle earlier and more massively than fast muscles. However, the 7 0 % - 8 0 % centralized nuclei observed in both slow and fast muscles indicated that regeneration occurred to the same extent in both muscle types (Coulton et al. 1988; Dangain and Vrbovfi 1984), regardless of their utrophin content.

Discussion

In our present work we correlate the expression of utrophin and the expression of developmental MHC as an index of an active regenerating process following necrosis. The dystrophic activity of mdx mouse has

been extensively studied but using various criteria such as histological and physiological parameters, connective tissue proliferation, number of centronucleatcd fibers, number of necrosed fibers, and expression of developmental MHC (Dangain and Vrbovfi 1984: "Fanabe et al. 1986; Tores and Duchen 1987, Woo et al. 1987; Carnwath and Shotton 1987; Coulton et al. 1988, Cullen and Jaros 1988; Karpati et al. 1988; McArdle et al. 1991; Dimario et al. 1991; Stedman et al. 1991; Weller et al. 1990; Dupont-Versteegden et al. 1992). Some discrepancies emerge from these studies. Our results, describing an acute phase of muscle necrose/regeneration becoming less conspicuous later in life, are at variance with those of Torres and Duchen (1987) and Karpati et al. (1988) but confirm and extend those of Dangain and Vrbovfi (1984), Carnwath and Shotton (1987) and Dimario et al. (1990). Utrophin was present at the periphery in all mdx mouse muscle fibers during the first postnatal period with a subsequent decrease after 2 weeks and it only disappeared from all muscles at 3 weeks. In normal mice Koga (1993), using polyclonal antibodies and quantification by Western blot, reported a decrease in the utrophin in fetuses from 13.5-day embryo to nearly none expressed at birth. The discrepancy in the timing of this decrease and our results may be due to the different techniques a n d / o r tools used, unless the disappearance of utrophin in mdx mouse was delayed in comparison to what occurred in normal mice. Only a comparison of normal and mdx mice in the same conditions could resolve this question. Nevertheless in mdx mouse muscle we noted that utrophin decreased and became undetectable at 3 weeks in fast and in slow muscles, and was present in larger amounts in adult slow muscles. Therefore the observed utrophin was reexpressed rather than not suppressed. The suggestion that the newly regenerated fibers do not degenerate again thanks to utrophin (Weller et al. 1990: Takemizu et al. 1991) is not coherent with the following two facts: observed expression of utrophin in severely affected patients (Voit et al. 1991) and, in the present study, the observation that the relative amount of utrophin present is independent of the severity of the pathology. Fast muscles showed more progressive attack with lower amounts of utrophin and the diaphragm, the most affected mdx mouse muscle (Stedman et al. 1991: Dupont-Versteegden et al. 1992). contained most of the utrophin. Utrophin may be reexpressed in regenerated mdx mouse fibers as part of the fetal program with no correlation with developmental M H C expression, as observed in humans (Helliwell et al. 1992). Moreover, a massive degenerative-regenerative process was concomitant with a decrease in utrophin only in the soleus. Fast muscles did not degenerate as suddenly as slow muscles but, in adult mdx mice with no apparent utrophin, fast muscles as well as slow muscles

II~0

with higher amount of utrophin, werc relatively rcsistant to further necrosis.Various factors may explain the diffcrential vulnerability of diffcrcnt muscle fiber to nccrosis. Cullcn and Jaros (1988) suggcstcd that thc widcsprcad myofiber necrosis occurring at 3 - 4 weeks in md,v mice is iriggcrcd by incrcascd mechanical demand which is grcatcr in slow thari in fast muscles, At this timc, ihc lack of dystrophin might become critical with the disappearance of utrophin in the soleus. This did not conflict with the obscrvations of Karpati ct al. (19H8) showing that small-caliber fibers do noi suffcr of necrosis. Neverthcless more frequently endured mechanical stress in the soleus and diaphragm muscles may also cxphtin the vulncrability to thc necrosis of thcsc muscles in spite of larger amounts of utrophin. In this optic, thc works of Weller ct al. (199(tl and Dick and \,rbowi (1993) also dcscribc a deterioration of md.v musclc induced by ovcrload. Othcr diffcrcnccs bctwccn mousc and human dyslrophin dcficicncy werc also noted in lhcse studics. First the degenerative proccss affects fast muscles less l h a n slow mtiseJcs whilc the revers( has bccn shoran in humans (Wcbsier ct al. 1988: Minctti ct al. 1991: Mar\n\ ct al. 1992). Sccondly, development is absolutcly normal in #tuLv micc, whcrcas in humans duririg prcclinical slages of Duchennc muscular dystrophy histopathological abnormal\tics have bccn observed (Bradley et al. 1972; Hudgson ct al. 1967). Thc potcntial inw4vcmct~t of other substitution proteins or myogenic factors should flow bc invcstigated in md.v mice, ( ' X M D dogs. olhcr dystrophin deficient models and in I)MD patients Io dctcrmine why the rcgcncration proccss is succcsshil in md_v micc. This approach could providc chics for ftittirc it~erap}. ?icknovtledgmcnls ~%c arc grcltclul Io I)i. T. Partridge, Dr. J. Moi~111 ([,oildoil, i r K ) 4Ild ,,%,. Scbillu' {Paris) 1]}I thuir help and for lilt' ,eil'l of md.~ ill\c(.. \Vu lhank l)r. l loclnlla h)r Jaboral~.)l'y Iacililics. M. to' ('tllltl. M Alloal alld J,().('. l,dgcl h~r the anlibodics, and M, M o r e l for help \\ith ihc ph(~iogral~hs. This \~ork ~alllt ~ Pul~liclUC
References , \ u g i c r , N.. B o u c l a u l ,1., Ldgcr. ,1.().('., Anoal, M., Nicholson, L.V.B., VIsclkcl M.A.. I d g c r .I..1. and J.F. Pcllissicr (1092) a honloh)guc ~51 dystiophin is cxpIcsscd at the blood vessel rncnlbrane of I ) M I ) and B M I ) palicnts: inlrllunological evidence. ,I. Neuro]. Sci.. 11)7: 233 23N. lllau, t1., W c b s l c r , ('. and (i.K. Pavlalh (10N3) Defective myolnlasl~ idcnlificd in I)MI). l'roc. Nail. Acad. Sci. USA, 811 (15): 4N5
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