Distinct roles for laminin globular domains in laminin alpha1 chain mediated rescue of murine ...

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Distinct roles for laminin globular domains in laminin alpha1 chain mediated rescue of
murine laminin alpha2 chain deficiency.
Gawlik, Kinga; Åkerlund, Mikael; Carmignac, Virginie; Elamaa, Harri; Durbeej-Hjalt,
Madeleine
Published in:
PLoS ONE
DOI:
10.1371/journal.pone.0011549
Published: 2010-01-01
Link to publication
Citation for published version (APA):
Gawlik, K., Åkerlund, M., Carmignac, V., Elamaa, H., & Durbeej-Hjalt, M. (2010). Distinct roles for laminin
globular domains in laminin alpha1 chain mediated rescue of murine laminin alpha2 chain deficiency. PLoS
ONE, 5(7), [e11549]. DOI: 10.1371/journal.pone.0011549
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Distinct Roles for Laminin Globular Domains in Laminin
a1 Chain Mediated Rescue of Murine Laminin a2 Chain
Deficiency
Kinga I. Gawlik, Mikael A˚kerlund, Virginie Carmignac, Harri Elamaa¤, Madeleine Durbeej*
DepartmentofExperimentalMedicalScience,MuscleBiologyUnit,UniversityofLund,Lund,Sweden
Abstract
Background:Laminina2chainmutationscausecongenitalmusculardystrophywithdysmyelinationneuropathy(MDC1A).
Previously,wedemonstrated thatlaminin a1chainamelioratesthediseaseinmice.Dystroglycan andintegrins aremajor
lamininreceptors.Unlikelaminina2chain,a1chainbindsthereceptorsbyseparatedomains;lamininglobular(LG)domains
4andLG1-3,respectively.Thus,thelaminina1chainisanexcellenttooltodistinguishbetweentherolesofdystroglycan
and integrins inthe neuromuscularsystem.
Methodology/PrincipalFindings:Here,weprovideinsightsintothefunctionsoflaminina1LGdomainsandthedivisionof
theirrolesinMDC1Apathogenesisandrescue.Overexpressionoflaminina1chainthatlacksthedystroglycanbindingLG4-
5domainsina2chaindeficientmiceresultedinprolongedlifespanandimprovedhealth.Importantly,diaphragmandheart
muscles were corrected, whereas limb muscles were dystrophic, indicating that different muscles have different
requirementsforLG4-5domains.Furthermore,theregenerativecapacityoftheskeletalmuscledidnotdependonlaminin
a1LG4-5.However,thisdomainwascrucialforpreventingapoptosisinlimbmuscles,essentialformyelinationinperipheral
nerveand important forbasement membraneassembly.
Conclusions/Significance:Theseresultsshowthatlaminina1LGdomainsandconsequentlytheirreceptorshavedisparate
functions inthe neuromuscular system. Understanding theseinteractions could contribute to design and optimization of
futuremedical treatment forMDC1Apatients.
Citation:GawlikKI,A˚kerlundM,CarmignacV,ElamaaH,DurbeejM(2010)DistinctRolesforLamininGlobularDomainsinLaminina1ChainMediatedRescueof
MurineLaminina2ChainDeficiency.PLoSONE5(7):e11549.doi:10.1371/journal.pone.0011549
Editor:AntoniL.Andreu,HospitalValld’Hebron,Spain
ReceivedMay12,2010;AcceptedJune21,2010;PublishedJuly19,2010
Copyright: (cid:2)2010 Gawlik et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding: Funded by Association Francaise contre les Myopathies, Muscular Dystrophy Association, Anna-Greta Crafoord Foundation for Rheumatological
Research,GretaandJohanKockFoundationandAlfredO¨sterlundFoundation.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.
*E-mail:[email protected]
¤Currentaddress:DepartmentofMedicalBiochemistryandMolecularBiology,BiocenterOulu,CenterforCell-MatrixResearch,UniversityofOulu,Oulu,Finland
Introduction isoform expressed in muscle and peripheral nerve. Through
interaction with transmembrane receptors it regulates major
Congenital muscular dystrophy type 1A (MDC1A) is an functions of the neuromuscular system and provides structural
autosomal recessive disorder caused by mutations in the gene supporttomusclefibers[6].Inmuscle,atleasttwodistinctprotein
encodinglaminin(LM)a2chain.Thegeneralclinicalhallmarksof complexes are known to be the key receptors for LMa2 chain;
MDC1A include neonatal onset of muscle weakness, hypotonia dystroglycanandintegrina7b1.Theirimportanceisunderscored
often associated with joint contractures, inability to stand and by the fact that absence of integrin a7 chain, as well as
walk,elevatedlevelsofcreatinekinase,whitematterabnormalities hypoglycosylation of a-dystroglycan cause various forms of
and dysmyelination neuropathy. Histological changes of muscles congenital muscular dystrophy [7,8]. Furthermore, different
comprisefibersizevariability,massivedegenerationandextensive studies involving manipulation of the dystroglycan gene in mice
connective tissue infiltration. Most patients die as teenagers since revealed an important role for dystroglycan in skeletal muscle
there is no treatment for this devastating disease [1]. Several [9–11]. Several studies indicated that the function of integrin a7
mouse models for MDC1A exist (e.g. generated LMa2 chain subunit and dystroglycan, being a part of the dystrophin-
mutants dy3K/dy3Kand dyW/dyW and the spontaneous mutant glycoprotein complex, could overlap [12–14]. However, recent
mouse strain dy/dy) and they adequately mirror the human studiesshowthatwhereasbothdystroglycanandintegrina7chain
condition [2–4]. contribute to force-production of muscles, only dystroglycan
LMs are extracellular proteins formed by a, b and c chains. contributes tothepreservation of sarcolemmalintegrity [15].
Together with other extracellular matrix components LMs form LMa2 chain receptors present in peripheral nerve include
specializedextracellularmatricescalledbasementmembranes[5]. dystroglycan, integrins a6b1, a7b1 and possibly integrin a6b4
LM-211 (composed of a2, b1 and c1 chains) is the major LM [16,17]. Dystroglycan, b1 and b4 integrin subunits have been
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LamininreceptorsinMDC1A
shown to be important for different aspects of myelination and cDNA (dE3LMa1). An in frame deletion between nucleotides
morphology of peripheral nerves, as revealed by conditional 8248–9289 (corresponding to LG4-5 domains) was accom-
disruptionoftheirgenesinSchwanncells[18–20].Thus,LM-211 plished by DraIII-SmaI restriction cutting and fusion of an
isacentralplayerlinkingthesereceptorsandtheirfunctionsinthe XhoI site with a BglII site. This DNA was cloned into the
neuromuscular system. pCAGGS vector [25], containing a CMV enhancer and a b-
LMa1 chain also binds to dystroglycan, integrin a6b1 and actin promoter.
integrin a7b1 (and perhaps integrin a6b4) [17,21–24]. Yet, it is
not expressed in the neuromuscular system [25]. We have Transgenic animals
previously explored the possibilities of paralogous gene therapy
Transgenic mice were generated by microinjections of
for MDC1A and demonstrated that LMa1 chain is an excellent
transgene DNA into the pronucleus of fertilized single-cell
substituteforLMa2chaininmurinemuscle,peripheralnerveand
C57BL/CBA embryos (Lund Transgenic Core Facility, Lund
testis [25–28]. Additionally, LMa2 chain deficiency leads to
University,Sweden).MicecarryingdE3LMa1chainDNAwere
perturbed expression of integrin a7 subunit, and reduced
identified by PCR as described previously [25]. Positive
expression of the core protein of a-dystroglycan (but not a-
founders overexpressing truncated LMa1 chain in the neuro-
dystroglycan glycosylation), at the sarcolemma [29–31]. Notably,
muscular system (lines No. 3 and 4) were further bred with
LMa1chainoverexpressionrestoresintegrina7chainexpression, dy3K/+ mice [2], followed by sib breeding to generate LMa2
indicating that this receptor could be crucial for improvement of chaindeficientanimalsthatexpressdE3LMa1chain(dy3K/dE3
muscle functionindystrophicanimals [32]. mice). Dy3K/dy3K mice overexpressing full length LMa1 chain
The LMa1 and a2 chains bind dystroglycan and integrins by (dy3KLMa1 mice) were previously described [25–28]. Dy/dy
distinct domains. The a1 chain binds dystroglycan via its C-
mice used for heart studies were obtained from Jackson
terminal LG4 domain and integrin binding occurs via a1LG1-3
Laboratory.
[33,34].ThisisdifferentfromLMa2chainbindingwherethereis
considerable overlap in binding to dystroglycan and integrins.
Exploratory locomotion and body and muscle weight
Both a2LG4-5 and a2LG1-3 bind dystroglycan, whereas only
analyses
a2LG1-3 binds integrins [23,35]. The LMa1 chain can thus be
Exploratory locomotion was examined in an open field test. A
used more efficiently to distinguish between the roles of LM
mousewasplacedintoanewcageandallowedtoexplorethecage
binding to dystroglycan and integrins in the neuromuscular
for 5 min. The time that the mouse spent moving around was
system. Since LMa1 chain functions almost equally well as a2
measured. For all experiments, 10-week-old dy3K/dE3 animals
chainintheneuromuscularsystem,weusedthissubunitinorder
(n=16) were compared with10-week-old control mice (wild-type
to dissect the roles of the aLG domains and their receptors in
or dy3K/+) (n=8) and 5-week-old dy3K/dy3K mice (n=6). For
MDC1A pathogenesis and rescue. Hence, we produced and
weight analysis dy3K/dE3, control mice and dy3K/dy3K animals
characterized animals completely deficient in LMa2 chain, but
insteadoverexpressingatruncatedformofLMa1chain(dy3K/dE3 were sex- and age-matched (5-week-old) (n=14, n=3, n=11,
respectively, for females; n=8, n=4, n=8, respectively, for
mice) that lacks the dystroglycan binding site (LG4-5 domains at
males).Quadricepsandtibialisanteriormusclesfrom2-month-old
the C-terminus, also known as the E3 fragment), but retains the
wild-type (n=3), dy3K/dE3 (n=3) and 4-week-old dy3K/dy3K
integrin binding site(LG1-3,see Fig.1A) [33,34].
mice(n=4)wereusedtoestimatetheratioofwetmuscleweightto
body weight. Muscles from both legs were weighed and average
Materials and Methods
musclemasswascalculated.Unpairedt-testwasusedforstatistical
Ethics statement analysis.
All mouse experimentation was approved by the local (Lund
district)ethicscommittee(permitnumberM62-09).Allmicewere Creatine kinase activity
maintainedinanimalfacilitiesaccordingtoanimalcareguidelines. Blood was collected from the tail vein of 2-month-old control
mice(wild-typeordy3K/+)(n=10),dy3K/dE3(n=10)and4-week-
Transgenic construct old dy3K/dy3K mice (n=3) into EDTA-tubes and spun down two
Approximately1 kboftheC-terminalpartwasremovedfrom times for 5 minutes at 3500rpm. CK_P_S_cobas method was
mouse full-length LMa1 chain cDNA to generate truncated used by Clinical Chemistry Laboratory at Ska˚ne University
Figure1.GenerationofdE3LMa1transgenicanimals.(A)SchemepresentingLM-111structure.Full-lengthLMa1chainwithLG1-5domains
andtruncatedLMa1chain(dE3LMa1)withLG1-3domainsaremarkedtogetherwiththeirtransmembranereceptors.(B)Schematicpresentationof
transgenicconstructwithdenoted1kbdeletion(LG4-5).Restrictionsitesusedtoengineertheconstructareshown.
doi:10.1371/journal.pone.0011549.g001
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Hospital to quantify enzyme activity in plasma. Unpaired t-test Quantification of fiber size distribution, central
was usedforstatistical analyses. nucleation and fiber number
Diaphragmandlimbmusclesfromatleast3animalsfromeach
Histology and immunofluorescence microscopy group(4–6-week-oldwild-type,dy3K/dy3Kanddy3K/dE3mice)were
Skeletal muscle, heart, peripheral nerve and spinal roots analyzed.MinimalFeret’sdiameterwasmeasured[38]foratleast
cryosections(7 mm)fromcontrol(wild-typeordy3K/+),dy3K/dy3K, 2600 fibers for each group. The same number of fibers was used
dy/dy, dy3K/dE3 and dy3KLMa1 mice were either stained with for quantification of fibers with centrally located nuclei. An
hematoxylin and eosin or subjected to immunofluorescence additionalgroupof4–6-month-olddy3K/dE3animalswasincluded
analysis using following antibodies: rat monoclonal mAb200 for quantification of diaphragm fibers. Fibers from quadriceps
againstLMa1LG4domain[25],rabbitpolyclonal1057+against muscle from 4–6-week-old wild-type (n=3), dy3K/dy3K (n=3) and
LMa1 LN/LEadomain(N-terminus)(kindlyprovidedbyDr.T. dy3K/dE3 mice (n=3) were counted within a square of 646106
Sasaki) [36], rabbit polyclonal 1100+ against LMa4, (kindly pixels2.Unpaired t-testwas usedfor statistical analysis.
provided by Dr. T. Sasaki), rabbit polyclonal 1113+ against
LMa5 (kindly provided by Dr. T. Sasaki), rat monoclonal Treadmill exercise and Evans blue dye injection
MTn15 against tenascin-C [25], rabbit polyclonal U31 against Dy3K/dE3mice(n=4)wereexercisedfor30minonatreadmill
integrin a7B subunit (kindly provided by Dr. U. Mayer) [37],
Exer 6M (Columbus Instruments) at a downhill angle of 15u.
mouse monoclonal IIH6 against a-dystroglycan (Upstate Bio-
During the first 2 min the speed was gradually increased from
technology), mouse monoclonal F1.652 against embryonic
7 m/min up to 14–16m/min. Within 30min after completed
myosin heavy chain (Developmental Studies Hybridoma Bank,
exercise the mice were injected i.p. with Evans blue dye (EBD)
Iowa), rabbit polyclonal anti-collagen, type IV (Chemicon),
(Sigma Aldrich) dissolved in sterile saline (concentration: 0.5 mg
mouse monoclonal 46 against caspase-3 (BD Transduction
EBD/0.05ml saline; amount: 50ml per 10g body weight). After
Laboratories).Mouseonmousekit(Vector)wasusedforstaining
approximately24h,muscleswerecollectedandquicklyfrozenin
with embryonic myosin heavy chain according to manufacturer
liquid nitrogen. Unexercised mice were injected with EBD and
instructions. Tissues were fixed with 4% PFA at room
usedascontrols.Musclecryosections(8 mm)werefixedinice-cold
temperature (for laminin, tenascin-C, embryonic myosin heavy
acetone at 220uC for 10min, washed and mounted with
chain, collagen-IV and caspase-3 stainings), or with acetone at
FluorSave (Calbiochem). By fluorescence microscopy analysis,
220uC(forintegrina7B) or with 8%formaldehyde,followedby
EBDuptake into musclefibers was visualizedby redemission.
methanol at 220uC (for a-dystroglycan). Sections were analyzed
using a Zeiss Axioplan fluorescence microscope. Images were
Cardiotoxin injections
capturedusinganORCA1394ERdigitalcamerawithOpenlab
Tibialisanteriormusclesfrom6control(wild-typeordy3K/+),6
3 software. Images were prepared for publication using Adobe
dy3K/dy3K and 6 dy3K/dE3 mice were injected with cardiotoxin
Photoshop software.
(10 mmol/L in saline). Control and dy3K/dE3 mice were 2–3-
month-old. Dy3K/dy3K mice were 3-week-old. Three mice from
Immunoblotting
each group were sacrificed 4 days after injection and the other 3
ForLMdetectionproteinswereisolatedfrom100mgofdy3K/
after 11 days. Both injected and contralateral uninjected tibialis
dE3 and dy3KLMa1 muscles (3 mice from each group) by brief
anterior muscles were collectedand analyzed.
sonication in 1 mmol/L EDTA in TBS with 1:25 dilution of
protease inhibitors (Complete EDTA-free, Roche Diagnostics).
Electron microscopy and toluidine blue staining
For integrin detection proteins were extracted from 100mg
skeletalhomogenizedmusclepowderof3wild-typeanddy3K/dE3 Quadriceps femoris muscles, heart, diaphragm, sciatic nerves
mice in 1% Triton X-100, 50mM Tris-HCl, pH 7,4; 1mM andspinalrootsfromwild-type,dy3K/dy3Kanddy3K/dE3micewere
CaCl , 1mM MgCl and 1:25 dilution of Protease Cocktail fixed for 2 hours with 1.5% glutaraldehyde/1.5% paraformalde-
2 2
(Complete EDTA-free, Roche Diagnostics). Samples were incu- hyde, rinsed in So¨rensen’s phosphate buffer, post fixed in 1%
bated for 1 hour and spun down at 4uC. The supernatants were OsO4 and then embedded in Epon. Ultra thin sections were
collected and the protein concentration was determined using stained with uranyl acetate and lead citrate. Specimens were
BCA assay (Pierce). Dystroglycan was isolated using agarose examined by transmission electron microscopy (Philips CM 10).
bound wheat germ agglutinin (Vector) and N-acetyl-D-glucos- Three to4 animals fromeachgroup wereanalyzed.
amine (Sigma) as described before [32]. Lysates containing LM,
integrin and dystroglycan were separated on 5% or 8% Results
polyacrylamide-SDS gels under reducing or non-reducing condi-
Generation of dy3K/dy3K mice overexpressing dE3LMa1
tions.EHSLM(Invitrogen)wasusedasacontrolforLMblotting.
Proteins were transferred to nitrocellulose membranes (Amer- chain
sham). Membranes were blocked for 1 hour in 5% non-fat dry WehavegeneratedmiceoverexpressingLMa1chaindevoidof
milkin1xTBSwith0.02%Tween-20andincubatedovernightat LG4-5domains(comprisingtheE3fragment)underthecontrolof
4uC with a rabbit polyclonal antibody detecting LMa1LG3 a CMV enhancer and b-actin promoter (Fig. 1A and B) (dE3
domain (kindly provided by Dr. T. Sasaki); rabbit polyclonal mice), Mice overexpressing dE3LMa1 in skeletal muscle, periph-
antibodyagainstintegrina7B(kindlyprovidedbyDr.U.Mayer); eralnerveandheartweremaintained(transgeniclinesNo.3and
rabbitpolyclonalantibodyagainstb-dystroglycan[25]andmouse 4)(FigureS1,seealsoFig.2).TheexpressionoftruncatedLMa1
monoclonalantibodyIIH6 againsta-dystroglycan. Detectionwas chain was detected using antibodies against the N-terminal
performedwithECLkit(Amersham).ExpressionofLMa1chain, domains of LMa1 chain and the LG4 domain, respectively.
integrina7Bsubunit,a-andb-dystroglycanwasnormalizedtoa- Immunofluorescence staining with the antibody directed against
actininexpression(detectedwithmousemonoclonalantibodyEA- N-terminal domains of LMa1 chain demonstrated patchy
53, Sigma). Band intensity was measured using ImageJ software. expression of truncated LMa1 chain in basement membranes of
Unpaired t-testwas usedfor statistical analyses. skeletalandcardiacmuscle,andinendoneuriumandperineurium
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Figure2.ComparisonofexpressionlevelsofLMa1chainbetweendE3transgenicmice,dy3K/dE3anddy3KLMa1mice.(A)Truncated
LMa1chainisupregulatedinskeletalmuscle(SM),diaphragm(Dia),peripheralnerve(SN)andheart(He)indy3K/dE3micecomparedtodE3mice
expressingLMa2chain.Itreachesthelevelsoffull-lengthLMa1chainexpressionindy3KLMa1mice.Threeanimalsfromeachgroupwereanalyzed.
Bars,50mm.(B)Immunoblottingoftissueextractsfromwild-type,dy3K/dE3anddy3KLMa1skeletalmuscleandEHSextractwitharabbitpolyclonal
antibodyagainstLMa1LG3domain.Quantificationofsignalsrevealedthatthereisapproximately2.5-foldmoreoftruncatedLMa1chainindy3K/dE3
musclescomparedtofull-lengthLMa1chainindy3KLMa1muscles(p=0.0194).Resultsareshownasmeans6SEM.Theshiftinmolecularweightof
truncated(350kDa)vs.full-length(400kDa)LMa1chainbecameapparentafterrunningthesamplesforalongertime(datanotshown).
doi:10.1371/journal.pone.0011549.g002
ofsciaticnerveofdE3transgenicmice(FigureS1).Nostainingwas 4.5-fold upregulation of integrin a7B in dy3K/dE3 skeletal muscle
detected with the antibody directed towards LG4 domain, by immunoblottingexperiments (Fig. 3B).
indicating the overexpression of truncated LMa1 chain. Staining LMa2 chain deficiency does not significantly alter a-dystrogly-
withbothantibodieswasdetectedinLMa1TGmiceoverexpress- can glycosylation and b-dystroglycan expression at the sarcolem-
ing full-length LMa1 chain (Figure S1) (described in 25) and it ma [32], probably because other ligands (e.g. perlecan) are still
indicated a higher level and more homogeneous expression of present. By immunofluorescence analyses, expression of a-
LMa1 chain in these animals. Notably, overexpression of dystroglycan also appeared normal in dy3K/dE3 limb, diaphragm
truncated LMa1 chain in mice revealed no discernible patholog- and heart muscle and in sciatic nerve (Fig. 4A). In addition, we
ical phenotypes. quantifiedexpressionofa-andb-dystroglycanandtheyremained
Next,dE3micefromline3and4werefurthermatedwithmice thesame indy3K/dE3vs. control skeletal muscle(Fig.4B).
carryingthemutationinLama2gene(dy3K/+),tocreatemicethat All in all, these results suggest that integrin a7B is increased,
are devoid of LMa2 chain but instead overexpress dE3LMa1 whereas dystroglycans appear normally expressed in dy3K/dE3
chain (dy3K/dE3mice). muscles.
ExpressionoftruncatedLMa1chainisupregulatedupon Dy3K/dy3K mice with dE3LMa1 transgene have improved
LMa2 chain deficiency overall health
We analyzed the expression of dE3LMa1 chain in dy3K/dE3 Dy3K/dy3K mice completely deficient in LMa2 chain were
mice in a similar manner as in dE3 mice (only the staining with previously described [2]. Briefly, these animals suffer from severe
theantibodyagainstN-terminaldomainsisshown).Interestingly, muscle wasting, growth retardation, peripheral neuropathy and
upon LMa2 chain deficiency the truncated LMa1 chain was die approximately 3–5 weeks after birth. As shown in Fig. 5, the
upregulated in all examined tissues (skeletal muscle, diaphragm, overallhealthofdy3K/dE3micewasimprovedcomparedtody3K/
heart, peripheral nerve) compared to dE3 mice (Fig. 2A). Also, dy3K mice. First, dy3K/dE3 mice live longer. As demonstrated by
theexpressionlevelsseemedtoreachthosedetectedindy3KLMa1 the survival curve, approximately 75% of dy3K/dE3 animals
mice overexpressing full-length LMa1 chain. We also noted survive up to 3 months (Fig. 5B). Further estimation of dy3K/dE3
intracellularstainingoftruncatedLMa1chaininskeletalmuscle survivalencounteredobstacles.Duetohindlegparalysis,severalof
them were sacrificed according to the guidelines of the ethical
(Fig. 2A). Western blot analyses with an antibody against
LMa1LG3 domain revealed even higher expression (approxi- permit. Nevertheless, many dy3K/dE3 mice survive much longer
mately 2.5-fold) of dE3LMa1 chain in dy3K/dE3 muscles than 3 months.Our oldest animals diedoneyearafterbirth.
compared to full-length LMa1 chain in dy3KLMa1 muscles Second,dy3K/dE3animalsarebiggerthandy3K/dy3Kmice.At2
weeksofage,dy3K/dy3Kmicecanbeidentifiedduetotheirgrowth
(Fig. 2B). Therefore, we can rule out the possibility that the
observed phenotype of dy3K/dE3 mice described below is due to retardation whereas dy3K/dE3 mice appeared outwardly normal
(datanotshown).Furthermore,themajorityofdy3K/dE3animalsat
insufficient expression of truncated LMa1 chain. Also, it is clear
5 weeks of age can not be distinguished from normal littermates
that the regulatory mechanisms involved in LMa1 chain
(Fig. 5A). Weight gain for female and male dy3K/dy3K mice was
transgene expression are complex. We also assessed the
greatly delayed in 5-week-old mice whereas the weight gain for
expression of LMa4 and a5 chains. We and others have
female and male dy3K/dE3 mice was significantly increased
previously shown that expression of these two LM chains is
compared to dy3K/dy3K mice (Fig. 5C and data not shown).
upregulated in LMa2 chain deficient basement membranes
[25,39] (see also Figure S2). In dy3K/dE3 mice, the muscle However, dy3K/dE3 mice weigh significantly less than normal
littermates(Fig.5Canddatanotshown).Beginningfrom5weeksof
basementmembraneexpressionofLMa4anda5chainswasvery
age,thedifferenceinoverallphenotypebetweenmostofdy3K/dE3
similar to that of dy3K/dy3K mice (Figure S2). Hence, we suggest
andwild-typemicebecamemoreevident.Manydy3K/dE3animals
that the compensatory increase of LMa4 and LMa5 chains has
arevisiblysmallerthancontrollittermates(Fig.5A,middlepanel).
no beneficial effects in dy3K/dE3 muscles (which are analyzed in
However, some of the older dy3K/dE3 animals look outwardly
detail in the next paragraphs).
normal and are almost indistinguishable from their littermates
(Fig.5A,leftpanel).Also,theratioofquadricepsandtibialisanterior
Expression of integrin a7B and dystroglycan in dy3K/dE3 wet weight per body weight was similar in control and dy3K/dE3
tissues mice,whereastheratiowassignificantlyreducedindy3K/dy3Kmice
We next evaluated the expression of integrin a7B and (Fig.5Danddatanotshown).Hence,musclemasswasmaintained
dystroglycan in dy3K/dE3 muscles. Expression of integrin a7B is inproportiontothebodysizeindy3K/dE3mice.Nevertheless,most
reducedatthesarcolemmaofdy3K/dy3Klimbandheartmusclebut ofdy3K/dE3micedisplaysevereperipheralnerveabnormalities,as
to a lesser extent in dy3K/dy3K diaphragm (Fig. 3A). Notably, the demonstrated by temporary hindleg paralysis (either one or
expressionofintegrina7Bsubunitwasrestoredindy3K/dE3limb, occasionally two limbs) (Fig. 5A, arrow). When lifted by the tail,
diaphragm and heart muscle (Fig. 3A). Similarly, also full-length theyretracttheirhindlimbstowardthebody.Still,dy3K/dE3mice
LMa1 chain reconstituted integrin a7B chain at LMa2 chain perform much better in the locomotion activity test compared to
deficientsarcolemma[32].Wefurtherdetectedanapproximately dy3K/dy3Kanimals(Fig.5E),indicatingthatmusclefunctionislargely
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Figure3.Restorationandupregulationofintegrina7Bsubunitindy3K/dE3muscles.(A)Cross-sectionsoflimbmuscle(LiM),diaphragm
(Dia)andheartfromwild-type,dy3K/dy3Kanddy3K/dE3micewerestainedwithantibodiesagainstintegrina7B.Bars,50mm.(B)Immunoblottingof
totalproteinlysatesfromwild-typeanddy3K/dE3skeletalmuscleandquantitativemeasurementofintegrina7Bexpression.Thereisapproximately
4.5-foldmoreintegrina7Bindy3K/dE3muscle(p=0.0231).Resultsareshownasmeans6SEM.
doi:10.1371/journal.pone.0011549.g003
preserved.Yet,dy3K/dE3micemovesignificantlylessthancontrol DE3LMa1transgenereducesthedystrophicpathologyof
skeletal muscles and significantly prevents dystrophic
miceandthisissupposedlyduetothetemporaryparalysis(Fig.5E).
Finally, we noted that serum kinase activity was significantly changes in diaphragm and heart
elevatedindy3K/dE3mice(Fig.5F),indicatingthatmusclesmaybe We next examined the morphology of dy3K/dE3 skeletal and
dystrophic,despiteimprovedgeneralhealth. heartmuscle.Whenisolatingskeletalmusclesfromdy3K/dE3mice
In summary, survival during the first months of life and other (5-week-old and 4-month-old and older), it could be macroscop-
featuresoftheoverallphenotypeofdy3K/dE3micearenotgreatly ically seen that muscles were only modestly wasted (see also
dependent on LMa1LG4-5. Fig. 5D). However, histological analyses of muscle revealed vast
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Figure4.Normalexpressionofdystroglycansindy3K/dE3muscles.(A)Cross-sectionsoflimbmuscle(LiM),diaphragm(Dia),heartandsciatic
nerve(SN)fromwild-type,dy3K/dy3Kanddy3K/dE3micewerestainedwithantibodyIIH6againsta-dystroglycan.Bars,50mm.(B)Immunoblottingof
glycoproteinpreparationsfromwild-typeanddy3K/dE3skeletalmuscleandquantitativemeasurementofa-andb-dystroglycanexpression.Results
areshownasmeans6SEM.Nosignificantdifferenceinexpressionofa-andb-dystroglycanwasnotedbetweenwild-typeanddy3K/dE3muscle
(p=0.8200andp=0.7527,respectively).
doi:10.1371/journal.pone.0011549.g004
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Figure5.Overallphenotypeofdy3K/dE3mice.(A)5-week-olddy3K/dE3miceoftenhavenormalsize,remainalertandlivelywithgoodmuscle
tone.Awild-typeandady3K/dy3Klittermateareshownforcomparison.Withagethedifferencebetweenthebodysizeofnormalanddy3K/dE3mice
becomes more evident (middle panel). However some dy3K/dE3 animals (right panel) remain undistinguishable from littermates at older age.
Nevertheless,alldy3K/dE3micedevelopperipheralneuropathy(indicatedbyarrow).(B)Survivalcurvesofdy3K/dy3K(n=8)anddy3K/dE3mice(n=44)
upto3monthsofage.Curvesremainsignificantlydifferentfromeachother(p,0.0001).Around75%ofdy3K/dE3miceliveatleastupto3monthsof
age.(C)Wholebodyweightsof5-week-oldfemalecontrol,dy3K/dy3Kanddy3K/dE3mice.Bodymassispartiallyrecoveredinfemaledy3K/dE3mice.
Theyweighsignificantlymorethandy3K/dy3Kmice(p,0.0001),butsignificantlylessthancontrolanimalsp,0.0003).(D)Proportion(inpercentage)of
thewetweightofquadricepsmuscletothebodyweightincontrol,dy3K/dy3Kanddy3K/dE3mice.Comparedtocontrolmice,theratioisnormalin
dy3K/dE3(p=0.8001)butsignificantlyreducedindy3K/dy3Kmice(p=0.0003).(E)Exploratorylocomotionof10-week-oldcontrolanddy3K/dE3mice
and 5-week-old dy3K/dy3K mice. Dy3K/dE3 mice are significantly more active than dy3K/dy3K mice (p,0.0001) and less active than control mice
(p=0.0099).(F)Serumcreatinekinase(CK)activityincontrol,dy3K/dy3Kanddy3K/dE3mice.ThereisnodifferenceinCKactivitybetweendy3K/dy3Kand
dy3K/dE3mice,butdy3K/dE3remainsignificantlydifferentfromcontrolmice(p=0.0022)Eachbarrepresentsthemean6SEM.
doi:10.1371/journal.pone.0011549.g005
regenerationofmusclefibersinlimbmuscles,demonstratedbythe been shown to contribute to the severe dystrophic changes in
presence of small fibers with centrally located nuclei (Fig. 6A). muscles from MDC1A patients and LMa2 chain deficient mice
Approximately 35% and 25% of 4–6-week-old dy3K/dE3 quadri- [2,41,42]. In both dy3K/dy3K and dy3K/dE3 muscles either single
ceps and triceps muscle fibers, respectively, contained centrally caspase-3positiveapoptoticfibersweredetectedorapoptosiswas
located nuclei and the numbers of centrally nucleated fibers did morerobust(Fig.7B).Incontrast,themusclesfromLMa2chain
not differ significantly from dy3K/dy3K muscles (data not shown). deficientmiceoverexpressingfull-lengthLMa1chain(dy3KLMa1)
The number of fibers in randomly selected areas was similar in were free of apoptotic fibers (no caspase-3 staining was observed,
wild-typeanddy3K/dE3quadricepsmuscle,butwithatendencyof Fig. 7B). Interestingly, apoptosis did not take place in dy3K/dE3
more fibers in dy3K/dE3 mice (probably due to the presence of diaphragms,whereasapoptoticfiberswerepresentindiaphragms
smallregeneratingfibers).Interestingly,asimilarnumberoffibers from dy3K/dy3K mice (Fig. 7B). This data strongly suggests that
was also noted in dy3K/dy3K quadriceps muscle (Figure S3). LMa1LG4-5protectslimbmusclesfromapoptosis,mostprobably
However, average fiber diameter is smaller (data not shown) and via dystroglycan binding, whereas truncated LMa1 chain is
insteadmusclecontainsfibrotictissue(seeFigure8A).Thenumber sufficient toprevent apoptosis indiaphragm musclefibers.
offiberswithcentrallylocatedwasevenhigherinlimbmusclesof Regardless of apoptotic cell death, muscle replacement with
4-month-olddy3K/dE3animals,indicatingthatpathologyworsens connective tissue,so evidentin dy3K/dy3Kmice [25],was not very
overtime(Fig.6Aanddatanotshown).Nevertheless,theseresults obvious in dy3K/dE3 muscles (Fig. 6A). This tendency was also
indicate that dy3K/dE3 muscles undergo damage but that the demonstratedbytenascin-Cstaining.Tenascin-Chasbeenshown
constant regeneration and muscle mass is maintained with age. tobeupregulatedandextendstotheinterstitiumbetweenmuscle
Moreover, the diaphragm did not undergo degeneration/regen- fibers in dy/dy and dy3K/dy3K mice [25,43]. Some muscles from
eration cycles and its morphology appeared near normal in 5- different dy3K/dE3 animals showed moderate upregulation of
week-old and 4-month-old animals (Fig. 6A–C). Dy3K/dy3K tenascin-C(Fig.8A,twoindividualsareshown,fouranimalswere
diaphragm at 4–6-weeks of age displayed about 16% of analyzed). However, tenascin-C expression was less pronounced
regenerated muscle fibers with central nuclei. A significant than indy3K/dy3Kmuscles.Also,some dy3K/dE3limbmuscles did
reduction was found in dy3K/dE3 diaphragm, both in young and not display tenascin-C upregulation (Fig. 8A). Moreover, dia-
older animals and the numbers did not differ significantly from phragm did not showany signsof fibrosis (Fig.8A).
wild-type diaphragm (Fig. 6B). We also determined the muscle Cardiomyopathy is not a major feature of MDC1A [1].
fiber size in 4–6-week-old diaphragm muscle. The fiber size However, 2-month-old dyW/dyW hearts show infiltration of
distribution was shifted towards smaller fiber sizes in dy3K/dy3K connective tissue [44]. Dy3K/dy3K mice probably die too early in
animals,comparedwithwild-typemuscles.Notably,theshiftwas order to develop heart fibrosis (data not shown). Therefore, we
largely prevented indy3K/dE3muscles (Fig.6C). compared 5–6-month-old dy3K/dE3 hearts with hearts from 8-
To demonstrate functional benefit conferred by the truncated week-olddy/dy mice, whichshow massivefibrosis intheventricle
LMa1chainindiaphragm,wesubjecteddy3K/dE3micetodownhill wall(Fig.8B).Asdemonstratedbyhematoxylinandeosinstaining,
treadmill exercise and sarcolemmal integrity was evaluated by dy3K/dE3 hearts did not display any fibrotic lesions (Fig. 8). This
Evansbluedye(EBD)accumulation.Ithaspreviouslybeenshown trend was further confirmed by absence of tenascin-C staining
thatonlyoccasionalEBD-positivefibersarefoundindy/dymuscles (Fig.8B).
[40].Inagreementwiththeseresults,wealsodetectedafewEBD- In summary, LMa1LG4-5 domains are important for securing
positivefibersinunexerciseddy3K/dy3Kmuscles.Wealsoobserveda the mechanical stability of limb muscle fibers in LMa2 chain
fewEBD-positivefibersinunexerciseddy3K/dE3limbmuscles,but deficiency,mostprobablybybindingtodystroglycan.Interesting-
almost none in dy3K/dE3 diaphragm (Fig. 7A). While it was not ly, LMa1LG4-5 domains are not involved in improvement of
possibletoexercisedy3K/dy3Kanimals,dy3K/dE3limbmuscleswere diaphragm and heart muscle morphology, indicating that other
susceptibletoexercise-inducedsarcolemmalinjuryasevidencedby sites of LMa1 chain (most likely integrin a7b1 binding modules)
increaseduptakeofEBD.Interestingly,downhillrunninginduced areresponsibleforfunctionalreplacementofLMa2chaininthese
verylittledamageindy3K/dE3diaphragm(Fig.7A).AlthoughEBD muscles.
uptake in exercised dy3K/dE3 limb muscles varied, both between
animalsandopposinglimbswithinthesameanimal,thediaphragm Skeletal muscle regeneration is not impaired in dy3K/dE3
wasconsistentlyunaffected.Hence,truncatedLMa1chainprevents mice
exercise-induced injury in diaphragm but not in limb muscles, Since muscle regeneration seemed to be continuously main-
indicating that different muscles have different requirements for tainedindy3K/dE3limbmuscles(Fig.6A),wenextanalyzedtheir
LMa1LG4-5domains. regenerativepropertiesinmoredetail.Weinjected2–3-month-old
The phenomenon of progressive muscle fiber damage in the control, dy3K/dE3 mice and 3-week-old dy3K/dy3K tibialis anterior
limbswasfurtherunderscoredbycaspase-3staining.Apoptosishas with cardiotoxin to induce muscle damage and trigger muscle
PLoSONE | www.plosone.org 9 July2010 | Volume 5 | Issue 7 | e11549