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Research Article

Expression of mdr1 is required for efficient long termregeneration of dystrophic muscle

David Israelia,⁎, Simindokht Ziaeia, Bernard Gjataa, Rachid Benchaouira, Philippe Rameaua,Thibaut Maraisa, So-ichiro Fukadab, Masashi Segawab, Hiroshi Yamamotob,Patrick Gonina, Olivier Danosa, Luis Garciaa

aGenethon, CNRS UMR 8115, Evry, FrancebDepartment of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +33 160778698.E-mail address: Israeli@genethon.fr (D. Isr

0014-4827/$ – see front matter © 2007 Elsevidoi:10.1016/j.yexcr.2007.02.036

A B S T R A C T

Article Chronology:Received 13 November 2006Revised version received27 January 2007Accepted 1 February 2007Available online 1 April 2007

The mouse mdr1a and mdr1b genes are expressed in skeletal muscle, though their preciserole in muscle is unknown. Dystrophic muscle is characterized by repeated cycles ofdegeneration and regeneration. To explore the role of the mdr1 genes during muscleregeneration, we have created a triple knockout mouse lacking the mdr1a, mdr1b, and thedystrophin genes. The resulting ReXmice developed normally and were fertile. However, asadults, ReX had a higher proportion of degenerating muscle fibers and greater long-termloss ofmusclemass thanmdx. ReXmuscles were also characterized by a reduced proportionof muscle side population (mSP) cells, of myogenic cells, and a reduced capacity for muscleregeneration. We found too that mSP cells derived from dystrophic muscle are moremyogenic than those from normal muscle. Thus, in dystrophic muscle, the mdr1 gene playsan important role in the preservation of themSP and of themyogenic regenerative potential.Moreover, our results suggest a hitherto unappreciated role of mdr1 in precursor cells ofregenerating tissue; they therefore provide an important clue to the physiologicalsignificance of mdr1 expression in stem cells.

© 2007 Elsevier Inc. All rights reserved.

Keywords:MDR1mdxMuscleStem cellsRegeneration

Introduction

Only a very low rate of tissue turnover is required for theroutine maintenance of a healthy skeletal muscle in the adultmammal. However, a very high regenerative response can beactivated by muscle trauma and produces efficient muscleregeneration. In the adult, this is largely attributed to themuscle satellite cells [1–3].

Duchenne muscular dystrophy (DMD) is a muscledegenerative disease arising from loss of expression ofthe dystrophin gene and is characterized by reducedmyofiber mechanical resilience and therefore an acceler-

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ated myofiber breakdown. In young DMD patients themuscle fiber regeneration initially sustains conservation ofmuscle mass but this diminishes with increasing age [4]. Inthe mdx mouse, as in DMD patients, lack of dystrophinexpression results in myofiber breakdown, but unlike inDMD patients, muscle regeneration capacity is maintainedfor most of the life span and the overall clinical decline isslow [5–7].

In a previous study we documented an upregulation ofmdr1a expression and activity in mouse muscle precursorcells (MPC) that were treated by the FGF6 muscle growthfactor [8]. The MDR encoded proteins are ATP-dependent

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membrane transporters of many compounds, such asphospholipids, peptides, and various drugs. Induction ofMDR gene expression may result in the development ofcellular multidrug resistance, a syndrome which oftendevelops in cancer cells of patients subjected to anti-cancerdrug treatment [9]. The mouse mdr1a and mdr1b areencoded by two different genes that share approximately85% amino acid identity with each other and 80% with thehuman MDR1. Together, mdr1a and mdr1b have a similartissue distribution and substrate range to the single humanMDR1 gene [10]. p-GP (a collective designation of the proteinsencoded by mdr1a and mdr1b) is expressed on the apicalsurface of secretory epithelial cells in liver, small intestineand kidney, and in small capillary endothelial cells of theblood–brain and blood–testis barriers. p-GP is thought toprotect these vital organs by active exclusion of a widevariety of cellular toxins [10]. Additionally, p-GP is expressedin a wide variety of stem cells [11–15].

The mdr1ab(−/−) mice show no obvious developmental nortissue homeostasis abnormalities, but do have less thannormal ability to remove drugs from their tissues [10,16].Study of these mice demonstrated a possible role for mdr1 inthe protection of vital organs, but did not provide insights intoits biological role in stem cells.

Both mdr1a and mdr1b are expressed in mouse skeletalmuscles [8,17], and both themdr1a(−/−) and themdr1a/1b(−/−)mouse skeletal muscles showed perturbed pharmacokineticsfor the mdr1 substrate vinblastin, suggesting that the twogenes are active in mouse muscle [10,16]; however, no specificphysiological role has been proposed for mdr1 expression inmuscles.

Mdr1 expression and activity are induced in many tissuesby a variety of stress signals (reviewed in [18]), notably, in amyogenic cell line by FGF6, a muscle growth factor [8].Additionally mdr1 has an anti-apoptotic activity indepen-dently of drug application, by interfering with the caspase-dependent apoptotic pathway [19,20].

Because mdr1 is induced in regenerating tissues, isexpressed in stem cells, and has an anti apoptotic activity,we have hypothesized that induced expression of mdr1could play a role in a stem-cell-dependent muscle regenera-tion process.

To test this hypothesis we crossed the mdr1ab(−/−) andmdx mice, producing a triple knockout mouse for mdr1a,mdr1b, and dystrophin genes, designated “ReX”. This mouseshows reduction in mSP and MPC percentage, decreasedmuscle regeneration, increased muscle degeneration, and inthe long term, a severe loss of skeletal muscle mass. Ourresults support the hypothesis that mdr1 gene expression isneeded during chronic muscle regeneration for the preserva-tion of muscle precursor cells and for efficient long-termmuscle regeneration.

Materials and methods

Generation of ReX mice

Mdx mice were crossed with the mdr1ab(−/−) mice [10]converted to the FVB background (Taconic, NY). The same

cross-scheme was used in previous studies [21,22]. In thefirst cross, mdr1ab(−/−) male and mdx female were pairedand produced male offspring that are heterozygous formdr1ab(−/−) and hemizygous for dystrophin deficiency. TheF1-generation males were back-crossed with mdx femalesfrom the original mdx colony to give rise to 100%dystrophin mutant F2 offspring. These F2 mice were PCRscreened (primers identical to [23,24]) for heterozygosis tothe mdr1ab(−/−) genes, and the mdr1ab heterozygousdystrophic F2 mice were paired once more to produce theF3 generation. Among the F3 mice PCR screening was usedagain to identify mice homozygous for mdr1ab, to producethe mdx-c colony, and the homozygous for the absence ofthe mdr1ab genes, to produce the ReX colony. The mdr1aand mdr1b genes are closely located on the same chromo-some [10], and we never observed any meiotic segregationbetween them.

Mouse weight curves

Each group consisted of at least 17 mice and was composed ofat least 3 littermates from litters of 4–7 offspring, in order tominimize non-specific effects related to the littermate size andmaternal surveillance. Mice were weighed once a week duringthe first 3 months of life and once a month afterward. Growthcurve analyses were performed using a 3 parameter nonlinearmixed effect model [25]. Calculations and graphics wererealized with the R 2.0.0 program (R development core team).

Muscle immunofluorescence and histology

In the Evans blue study (N=6) mice were injected IP withEvans blue dye (Sigma) as described [26] and were killed 24 hlater. Images were acquired using a Leica MacroFluo™,combined with a manual 16:1 zoom system (Leica Micro-systems AG, Germany), and a Digital ORCA II ER CCD Camera(Hamamatsu Photonics, Japan). In the dystrophin revertantstudy, the dystrophin revertant fiber was stained with the P7rabbit antisera [27]. Serial images of entire transversalsections were taken (as described above) and revertant fibercounts were performed as a blind study on 3 different sectionsper muscle, using the Saisam™ software (Microvision instru-ments, Evry, France). Revertant percentage was calculatedrelative to total fibers/muscle, which were stained by anti-laminin antibody and were counted as above. mdr1 immu-nostaining was performed on a frozen transversal sections,using the anti-mdr1 C-19 goat antisera (Santa Cruz) and antigoat-HRP-DAB. Anti rabbit-alkaline phosphatase-Fast-Red(both systems of Dako) was used for the detection of thelaminin antisera.

Denervation–devacularization (DD) was performed essen-tially as described previously [7,28], in 12–14 month mice(N=7). Mice were killed 1 week after the operation andtransversal sections were cut from the widest part of bothcontrol and operated EDL. Muscles were stained by H&E andthe developmental myosin heavy chain MAB (NCL-MHCd,Novocastra, UK), and regenerated fibers were identified (todistinguish from surviving fibers) by their small diameter,basophilic color, and positive immunostaining with thedevelopmental MHC antibody.

Fig. 1 – mdx-c mice are heavier than ReX mice. The overallbodyweight difference is specific for skeletal muscles.(A) Mice where weighed once a week until the age of3 months and thereafter once a month until the age of17 months. The real mean weight±standard error of themean is plotted on themodel curves. The asymptoticweightsof Rex andmdx-c were significantly different in both genders(p≤0.0001), as tested using a nonlinear asymptoticmixed-effects model. (B) A collection of skeletal muscles andinternal organs from 10-month female mdx-c and ReX mice(N=6) were weighted. Plotted are the average organ relativeweights (normalized to the entire body weight). Significantlyheavier organs in mdx-c (p≤ 0.05, marked by *) are thegastrocnemius, gluteus, quadriceps, and the tibialis anteriormuscles, while brains and stomachs are significantly heavierin ReX mice. The liver relative weight was divided by 10 forvisual purpose.

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Semi quantitative RT-PCR

Essentially as described before [29]. Primer sequences appearin the supplemental information.

Extraction of muscle mononucleated cells (MMNC), Hoechststaining and cell sorting

Extraction staining and sorting ofMMNCweredescribedbefore[8,30], except that in the present study the Hoechst 33342concentration was 5 μg/ml.We routinely used 4mice (about 8–10 g skeletalmuscle tissue) of each strain in eachexperiment inorder to have enough material and to minimize the experi-mental variation related to mouse variability. The mSP gatingwas defined individually in each experiment by comparison tothe Verapamil treated control MMNC in a way that less than0.01% of the Verapamil-treated cells was included within themSP gate. ThismSP gatewas divided in some experiments intoproximal and distal mSP in a 1 to1 proportion in the MMNC ofthe mdx-c muscles. Identical gating was used for the analysisof MMNC of all other strains. At least 250,000 events wererecorded and analyzed in each experiment.

Myogenic potential of mSP cells

Typically 80,000 mSP were plated immediately after sorting ina matrigel (BD bioscience)-coated 19 mm TC plate in muscledifferentiation media (DMEM, 2% horse serum, 50 mM HEPES,10 μg/ml Insulin, 10 μg/ml Apo-Transferin, Streptomycin, andPenicillin). After 1 week, cells were fixed in methanol andstained with desmin MAB (Sigma, clone U-10), followed by arabbit anti-mouse HRP and a DAB solution (DAKO) and werecontra-stained with propidium iodide directly in the PBS. Forthe desmin quantification, each 19 mm plate was entirelycovered by 120 images taken with the 4× objective, using theNikon E600 microscope with a motorized support stage. TheCartograph™ and Histolab™ programs (Microvision Instru-ments, Evry, France) were used for the photomontage and dataanalysis.

FACS analysis of total MMNC

For desmin quantification, MMNCwere fixed in coldmethanoland double stained for desmin (Sigma, clone U-10) and CD45(BD Bioscience). Cells were analyzed by FACScalibur andCellQuest software (BD bioscience) and the proportion ofdesmin positive (desmin +ve), CD45 negative (CD45 −ve) cellsout of the entire CD45 −ve population was analyzed bycomparison to the appropriate desmin isotype control.

Myogenic potential and quantification of SM/C-2.6 +ve MMNC

Freshly isolated MMNC were double stained with MAB SM/C-2.6 [31], and anti-CD45 antibody (BD Bioscience). Analysis andsorting were performed on a dual-laser MoFlo flow cytometer(Cytomation and Co). Sorted cells were plated on matrigel in amuscle differentiation medium (DM) for 1 week and imageswere taken using an inverted Zeiss microscope withNomarsky optics. For quantitative analysis, MMNC weredouble-stained and analyzed by FACS as above. The propor-

tion of SM/C-2.6 +ve and CD45 −ve cells was calculated relativeto the entire CD45 −ve population (N=3).

Results

Generation of ReX mice

Mdxmicewere crossedwith themdr1a/1b(−/−) [10] to generatea triple knockout mouse for the mdr1a, mdr1b, and thedystrophin genes (see details in experimental proceduresand Supplemental Fig. 1). The resulting mdr1a/1b(−/−)/mdxmice were designated “ReX”, while the mdr1a/1b(+/+)/mdxlittermate mice were designated mdx-control (mdx-c). Themdx-c, like the classical mdx mouse, have normal mdr1a andmdr1b genes expression and a mutated dystrophin.

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A reduced body weight in ReX compared to mdx-c is restrictedto skeletal muscles.

At first sight ReX mice showed no developmental abnormal-ities and were fertile. Body weight analysis, however, revealedsignificant differences between the ReX and mdx-c micebeyond the age of about 8 weeks (Fig. 1). At 160 days malemdx-c reached their maximum weight of 38.55 g on average,while ReX weighed only 34.15 g. Similarly, at 190 days thefemalemdx-c reached theirmaximal averageweight of 32.48 gwhile the ReX female weighed only 30.19 g. The growth curvesof Rex and mdx-c were significantly different in both genders

Fig. 2 – Increased proportion of necrotic myofibers in ReX musclEvans blue dye staining of the gastrocnemius in 10monthmdx-c (and ReX (D). Hematoxylin–eosin staining of a gastrocnemius of 6

(p≤0.0001). To determine if the reduced bodymass in the adultand old ReX compared to mdx-c mice is restricted to skeletalmuscles, we weighed a collection of skeletal muscles andinternal organs from 10month-old mdx-c and ReXmice (N=6)and calculated their relative mass (normalized to the totalbody mass; Fig. 1B). Most of the studied skeletal muscles weresignificantly smaller in ReX than in mdx-c mice. The largestweight difference was found in the gluteus (41% heavier inmdx-c) muscles. By contrast, the relative masses of non-muscle organs (kidney, liver, brain and spleen) were similar inthe two strains, or even significantly higher (brain andstomach), in ReX mice. These results suggest that body mass

es. (A) Histological studies in muscle transversal sections.A) and ReX (B), and of the tibialis anterior in 6monthmdx-c (C)-month old mdx-c (E and G) and ReX (F and H) mice.

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loss in adult and old ReX compared to mdx mice is particularto skeletal muscles.

Increased myofiber degeneration and reduced number ofmyofibers in ReX compared to mdx-c skeletal muscles

Skeletal muscles from newborn and young ReX mice werehistologically indistinguishable from those of mdx-c or the

classical mdx mice. In agreement with numerous studies ofmdx mice, we observed normal muscle architecture in allthree mouse strains until about postnatal day 20. Thereafter astrong wave of fiber degeneration/regeneration was observedboth in mdx-c and ReX muscles, characterized by massiveinfiltration of inflammatory cells and the appearance ofcentral-nucleated replacement myofibers (not shown). Histo-logical differences between ReX and mdx-c were, however,

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observed in adult and old mice. In skeletal muscle of adultmdx-c mice, in agreement with previous studies [5,7,32], onlyfew isolated fibers showed loss of membrane integrity andentry of Evans blue dye (EBD), while in ReX in 3 out of 6 micemore than 18% of the gatsrocnemius transversal sectionsurface was occupied by EBD-stained fibers (Figs. 2a–d).Interestingly, EBD-stained ReX myofibers appeared some-times in large patches (Fig 2d), rather than the sporadicdispersed staining pattern seen in mdx-c muscles (Figs. 2a, c),in somemuscles occupying up to 1/4 of the entiremuscle area,thus may represent zones of non-reversible muscle degenera-tion, leading eventually in ReX mice to a loss of muscle mass.Indeed, large areas of necrotic fibers in ReXmuscles were seenby H&E staining (Figs. 2f, h). Morphometric analysis oftransverse sections of gastrocnemius muscles in 6 month-old mice (N=6) showed significant differences in the popula-tion profiles of fiber areas betweenmdx-c, ReX, and the C57BL/6 mice (Supplemental Fig. 2). The average number of fibers/muscle inmdx-c (10370) was significantly higher (p≤0.05) thanin C57BL/6 (5757) and higher, slightly below significance(p≤0.064) compared to ReX mice (6574). Taken together,these results suggest that a high rate ofmyofiber degenerationcontributes to themuscle loss of weight that characterizes ReXmouse skeletal muscles.

Reduced regeneration in ReX skeletal muscle

Dystrophin positive “revertant”muscle fibers can occasionallybe detected inmuscles of DMD patients and of mdxmice (Figs.3A, a, b), resulting apparently from spontaneous alternativesplicing events in the dystrophin genes. The accumulation ofrevertant fibers in themdxmuscle serves as cumulative indexof muscle regeneration [33]. We have identified significantlyhigher percentages of revertant fibers in quadriceps muscle of17-month mdx-c than of similarly aged ReX mice (N=4,p≤0.006), suggesting more extensive muscle regeneration inthemdx-c than in the ReXmicemuscles (graph in Figs. 3A). Tocompare directly the acute, short-term muscle regenerationpotential in ReX and mdx-c mice, we took advantage of thedevascularization denervation (DD) muscle damage model[7,28]. DD in the EDL muscle was studied in 1 year old ReX andmdx-c mice. Muscles analyzed 1 week after DD showedsignificant differences between the 2 strains (N=7). Oneweek after injury of EDL muscle, the regenerating area in themdx-c mice muscle was composed mainly of newly formed

Fig. 3 – Muscle regeneration is less efficient in ReX than in mdxquadriceps muscle of 17-month ReX and mdx-c (N=4). Muscle crpanels) or laminin (lower panels) antibodies. Dystrophin +ve revlaminin stained section. Total number of fibers per muscle was crespectively) and the percentage of revertant (dystrophin +ve) fiband ReX, respectively), as presented in the graph. (B) EDLmusclesa 1-year old ReX (a, c, and e) and mdx-c (b, d, and f) (N=7). Muschematoxylin–eosin. The contra-lateral (non-operated) EDL (a andmyotubes (marked by an arrow, characterized by a small diametmagnification images (f, corresponding to the internal frame in d),to the internal frame in c) onlymyotubes that were not destroyed bhead), are visible, characterized by their large diameter, pink-pa

small myofibers (Figs. 3B, d, f). In contrast, the regeneratingarea in the equivalent muscles of ReX mice contained onlyvery few newly formed myofibers (Figs. 3B c, e), representingan inhibition or perhaps a delay in muscle regeneration. AMann–Whitney test of the proportions of regenerating muscle1 week after injury showed a significant difference betweenthe two strains (p≤0.029). These results show that EDLregeneration after DD is less efficient in ReX than in mdx-cmice.

Comparable vascularization in ReX and mdx-c muscles

Expression of mdr1 has been identified in some tissues inthe vascular endothelium [10]. Skeletal muscle is a highlyvascularized tissue. Muscle homeostatic maintenance aswell as muscle regeneration is dependent on an appropriateblood supply. To assay for a possible reduced angiogenesisin muscle of the ReX mouse, we counted capillaries in thetibialis anterior (TA) muscle of 7-week old ReX and mdx-cmice (N=6). Transverse sections were stained with the endo-thelial marker CD31 (Supplemental Fig. 3). Capillary to fiberratio was similar between the two strains; while the capil-lary density was found to be slightly higher in the ReX thanin mdx-c muscles, suggesting that decreased angiogenesis isnot the cause of the reduced regeneration in the ReX muscle.

mdr1 is expressed by an interstitial muscle mononucleatedcells (MMNC) and MMNC of ReX mice have a reduceproportion of mSP cells

MMNC are the entiremuscle derivedmono-nucleated cells. Asso, this population composed of the satellite cells, the non-satellite MPC, and the non-myogenic muscle mono-nucleatedcells (fibroblast for example). Additionally the entire MMNCpopulation composed of two complementary sub-populationsof mMP and mSP cells. RT-PCR and immunostaining wereused in order to localize mdr1 expression within the muscle.MMNC were isolated from hind limb skeletal muscles ofC57BL/6 mice and gene expression was compared betweenpurified MMNC and the entire non-fractionated muscle,containing both MMNC and muscle fibers. The expression ofthe muscle differentiation marker MCK was limited to theentire muscle fraction and almost totally absent from MMNC(Fig. 4A). In contrast, mdr1a and mdr1b expression wasenriched in the MMNC in parallel with the expression pattern

-c. (A) Analysis of revertant (dystrophin +ve) fibers in theoss sections where stained with either dystrophin (upperertant fibers are identified by a red dot on the correspondingounted (10867±1627 and 8875±910 in mdx-c and ReXers was calculated (1.84%±0.66 and 1.15%±0.49 in the mdx-cwere damaged by devascularization and denervation (DD) in

les were harvested 1 week after damage and stained byb) were used as control. Notice the presence of younger, basophilic color and large nucleus) in the mdx-c highwhile in the ReXhighmagnification images (e, correspondingy the DD operation (survivingmyotubes,marked by an arrow

le color, and small nucleus.

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of pax7 and ABCG2 transcripts. The house-keeping geneGAPDH expressed to a similar level in all cases.

Anti-mdr1 antibody staining of the TA muscle in mdx-cmice revealed a rare population of mdr1-positive cellsbetween the muscle fibers in an interstitial position (Fig. 4B),as confirmed by double staining formdr1 and the basal laminaprotein laminin (Figs. 4B, c). Together these results suggestthat mdr1 expressed in skeletal muscle in interstitial mono-nucleated cells.

Stem cells from a variety of tissues and species can beidentified by the SP phenotype based on dual emissionanalysis of Hoechst stained cells [34,35]. It has been shownby several groups that the ABCG2/Bcrp1 gene is principally

responsible for SP phenotype in stem cells. However, mdr1activity was also associated with the SP phenotype [24,36–38],and we have recently shown that FGF6 overexpression inC2C12 and primary myoblasts resulted in increased mdr1aexpression and SP proportion [8]. In agreementwith Zhou et al.[24,38], we have detected similar proportions of SP in bonemarrow of all four mouse strains C57BL/6, mdr1ab(−/−) ReXand mdx-c mice (data not shown). Importantly, skeletalmuscle was different, containing a significantly smallerproportion of SP cells in ReX mice than in any of the other 3mice strains (representative results in Fig. 4C,N=6 for ReX andmdx-c, N=3 for C57BL/6 and mdr1ab(−/−)). The total mSP wassmaller in ReX than mdx-c (p≤0.01). Moreover, the cellulardistribution within the FACS SP gating was strikingly differentin the ReXmice.While the overall SP content was about half inReX mice compared with other strains, this difference wasseen mainly in the proximal-SP (pr-SP) whose percentage was3–6 fold smaller than in the other strains (Fig. 4C, table), whilethe distal-SP (di-SP) proportion in ReX mice was only slightlyreduced (about 25% reduction). Thus, the same gating thatproduced a 1:1 ratio of pr-SP versus di-SP inmdx-cmice, gave a1:5 ratio in ReX mice (prSP is smaller in ReX than mdx-c,p≤0.05). We reasoned that the reduced mSP percentage couldresult either from the lack of mdr1-dependent Hoechstpumping activity or the loss of cells co-expressing mdr1 andABCG2. The second scenario would predict reduced ABCG2expression in ReX compared to mdx-c MMNC. Using quanti-tative RT-PCRwe found, in fact, slightly increased, rather thandecreased ABCG2 transcript level in ReX compared to mdx-cMMNC (Supplemental Fig. 4). This argue in favor of acompensatory ABCG2 upregulation in mdr1ab(−/−) mice[37,39], and fits with the observation of slightly increasedmSP on themdr1ab(−/−) mice (Fig. 4C). It is therefore likely thatthe reduced mSP proportion in ReX-derived MMNC reflects

Fig. 4 – mdr1 is expressed inMMNC andMMNC derived fromReX mouse characterized by reduced mSP proportion.(A) Expression of mdr1a and mdr1b is enriched in musclemononucleated cells. RT-PCR was performed with RNAsamples extracted from MMNC or from the entirenon-fractionated muscle (designated muscle). A typicalresult from one mouse (N=4) is shown. (B) Upper panel: lightimaging of transversal section of tibialis anterior muscle ofmdx-c mouse was stained with anti-mdr1 antibody (redarrow) and propidium iodide (PI). Middle panel: PI imaging ofthe same field. Lower panel: co-staining of mdr1 (greenarrow) and the basal lamina protein laminin (in red color). (C)Reduced mSP proportion in ReX mice. The mSP profile wasstudied in mdx-c and ReX, C57BL/6 and mdr1ab(−/−) mice.Each experiment was performed with 10 g of hind-limbmuscles derived from 4 individual mice, using 5 μg/mlHoechst. In the upper part are shown typical results of oneout of three (in mdr1ab(−/−) and C57BL/6 strains) and 6 (in themdx-c, ReX strains) independent experiments. The mSPgating was divided into “proximal” and “distal” sub-SPregionswith a proportion of 1:1 in themdx-c. Average valuesare presented in the table. Notice the low proportion ofproximal SP in ReX mice.

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primarily the lack of directmdr1-dependent Hoechst pumpingactivity. Together, these results suggest that in dystrophicmuscle the mdr1 channels are partially responsible forHoechst dye efflux in MMNC and that under the experimentalconditions used here, it is these mdr1 channels that areparticularly involved in the preservation of the proximal SPcell population.

Myogenic potential of muscle SP cells

To study the myogenic potential of mSP derived from C57BL/6, mdx-c, and ReX muscles, the different cell populations

Fig. 5 – In vitro myogenic potential of mSP. (A) Sorting gate of mpanels was set in a way that less then 0.05% of “contaminated” cverapamil (lower panel). Sorted cells were plated (80,000 mSP cemuscle differentiation medium. (B and C) One week later, cells wiodide. Notice in B in the large magnification images that the moimages provide a complete view of the entire 19 mm tissue culturand methods) and the relative TC plate surface occupied by desmindependent experiments in each strain and are expressed in ar

were sorted and plated in muscle DM on matrigel as haspreviously been described [40]. Four mice of each strain wereused in order to collect enough mSP cells and minimize intermouse variation. In agreement with Meeson et al., weobtained in vitro myotube differentiation from the pure mSPcell cultures but with very few myotubes in the C57BL/6-derived mSP cultures. Surprisingly, myotube formation wassignificantly more efficient in mSP cultures derived from bothmdx-c and ReX mice. In contrast to C57BL/6 mSP, the mSPderived from dystrophic muscles attached rapidly (within48 hours) to the matrigel substrate and differentiated intomyotubes that sometimes even formed contractile networks

SP cells from mdx-c (a), ReX (b), and C57BL/6 (c) in the upperells could be identified in the presence of the mdr1 inhibitor,lls/plate), on matrigel coated-19 mm TC plates, directly inere fixed and stained with desmin antibody and propidiumnonuclated cells are desmin −ve. (C) The small magnificatione plates. (D) The photomontages were scanned (see Materialsin-positive myotubes was calculated. Results are from twobitrary units.

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(Figs. 5B, C). Quantitative analysis of the area occupied bydesmin positive myotubes in the entire tissue culture plate(N=2 independent plates for each strain) showed thatmyotube formation was in average about 8 times moreefficient in the cultures derived from dystrophic mSP (eitherReX or mdx-c) than those derived from the C57BL/6 mSP.These data indicate that mSP cells derived from dystrophicmuscles are distinctly more myogenic than those originatingfrom normal muscle.

Lower level of myogenic MMNC in ReX than mdx-c

We have used two different myogenic markers to comparethe myogenic potential of MMNC from ReX and mdx-c mice.The first, desmin, is an early differentiation marker of MPC[41,42]. From a double stain of MMNC for desmin and CD45followed by FACS analysis, we calculated the proportion ofdesmin-positive (desmin +ve) cells from the entire non-hematopoietic fraction, i.e. the CD45 negative cells (CD45 −ve),of the MMNC, finding a significantly higher proportion of

Fig. 6 – Reduced proportion of myogenic markers in ReX MMNCdesmin antibodies and analyzed by FACS. (B) MMNC from mdx-anti-mouse satellite cell SM/C2.6 antibody. Three distinct cell po(b) SM/C2.6 low and negative, CD45 negative, and (c) CD45 positiplated on matrigel in DM and were incubated for 1 week. (C) Thethe different MMNC populations in ReX compared to the mdx-c (Nand CD45 proportion is significantly lower (p≤0.02) in mdx-c tha

CD45 −ve, desmin +ve MMNC in mdx-c than in ReX mice(Fig. 6A). However, desmin staining required fixation of thecells and therefore their myogenic potential could not befurther validated in tissue culture. We therefore used a secondmyogenic marker, the SM/C2.6 antibody that recognizesspecifically mouse muscle satellite cells [31]. To confirm themyogenic nature of the SM/C2.6 +ve cells derived fromdystrophic muscle, we double stained mdx (in Fig. 6B) andReX (not shown)MMNCwith CD45 and SM/C2.6 antibodies andFACS-sorted accordingly threedifferent cell populationswhichwere plated on matrigel in DM. Only the CD45 −ve SM/C2.6“bright” cells formed myotubes, while the double negative orthe CD45 +ve cells (either SM/C2.6 +ve or −ve) did not,confirming the specificity of this antibody combination forMPC. The same staining protocol was employed to comparethe SM/C2.6 level in mdx-c versus ReX MMNC (fig 6C). Thepercentage of CD45 −ve, SM/C2.6 “bright” cells in the entirenon-hematopoietic fraction (CD45 −ve) of the MMNC wassignificantly (p≤0.04) lower in ReX MMNC than those frommdx-c. Additionally, we have identified a higher proportion of

. (A) ReX and mdx-c derived muscle MMNC stained withc and ReX mice were double-stained with CD45 and thepulations were sorted: (a) SM/C-2.6 “bright”, CD45 negative,ve. The sorted cells (derived from mdx-c mouse in a–c) weresame staining protocol was used to analyze the proportion of=3). SM/C-2.6 +ve proportion is significantly higher (p≤0.04)

n in ReX.

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CD45 +ve cells in ReX muscles (p≤0.02). Together, our resultssuggest that in the absence of mdr1 dystrophic muscles ofadult mice have a low proportion of mSP, and furthermore,that mSP cells derived from dystrophic muscle (of both mdx-cand ReX) have a higher myogenic potential than mSP cellsderived from normal muscle (of C57BL/6), and lastly, thatMMNC derived from ReX possess reduced myogenic potentialcompared to MMNC derived from mdx-c.

Discussion

Muscle pathology in the mdx mouse is characterized by mildclinical symptoms and an almost normal life span. The exactnature of themolecularmechanisms that enable such effectivemuscle preservation is still largely unknown. However, invol-vement of putative muscle stem cells in persistence of muscleregeneration in the mdx mouse may be critical [43,44].

We have recently identified and sorted SP cells fromcultured C2C12 and primary myoblasts. These cells possessedsome features in commonwith stem cells in addition to the SPphenotype, including high proportion of cells arresting in G0and low expression of tissue-specific transcription factors [30].These SP cells expressed several members of the ABC-transporter family, including mdr1a, mdr1b, and ABCG2.Because mdr1 is expressed in stem and progenitor cells,induced in regenerating tissues and has an antiapoptoticactivity, we hypothesized a role for mdr1 during muscleregeneration, possibly in the protection of muscle progenitorcells. To experimentally test this hypothesis, we have crossedthe mdr1a/1b deficient mouse with the mdx mouse. Inagreement with our expectation, the resulting ReX mouseshows more severe muscle pathology than the mdx-c mice.

Influence of mdr1 expression on body and muscle mass indystrophic mice

No major differences were observed between the two strainsup to the age of two months, after which, however, the ReXmice grew more slowly. Reduced weight in ReX mice wasrestricted to the skeletal muscles. The relative (but not theabsolute) weights of some of the non-muscle organs wereeven significantly higher in ReX than mdx-c mice, due to thereduced total body mass of the ReXmice. While the total bodymass reduction of ReX mouse was moderate, the loss of massin muscles was drastic and reached more than 35% in thequadriceps and gluteus muscles.

Histological analysis confirmed that the skeletalmuscles ofReXwere not only lighter but also contained fewer fibers and ahigher proportion of necrotic one, compared to mdx-c.Excessive myofiber degeneration and a loss of weight in ReXmuscles may be the outcome of a long-term complex process.Clearly, however, a reduced muscle regeneration capacitycould contribute significantly to this phenotype, a viewsupported by the lower numbers of small-sized fibers (thatmay represent young fibers) in the gastrocnemius of ReXcompared to mdx-c. Two methods have been used to identifydifferences in the efficiency ofmuscle regeneration in ReX andmdx-c. In the first approach,we have calculated the number ofrevertant fibers in the muscles of old mice as a reliable

cumulative index of the long-term regeneration of dystrophicmuscles [33], and found a significant higher revertantpercentage in mdx-c muscles. In the second approach, wehave compared the short-term regeneration capacity of themdx-c and ReX mice by using the DD regeneration assay,which, in our hands is more reproducible than myotoxininjection. The two methods are profoundly distinct andcomplementary; the first providing retrospective reflectionof a long-term chronic process, the second indicates of theshort-term acute muscle regeneration process. Both con-firmed that muscle regeneration is reduced in ReX comparedto mdx-c, thus explaining the lower muscle mass in ReX. Asecond cause of the loss of muscle mass in ReX is the fact thattheir muscle showed a distinctly higher rate of muscledegeneration than their mdx-c littermates. This does notseem explicable in terms of a direct effect of lack of MDRfunction, since mdr1 is expressed principally in MPC ratherthan myofibers, but it is in line with a number of observationsof a protective effect onmyofiber integrity associatedwithMPCoveractivity, e.g. inmdxmice overexpressing IGF-1 or in whichmyostatin activity is lost or blockaded [45,46]. Ours is theinverse case,where loss ofmyogenic activity is associatedwitha reduction of myofiber resilience. In neither case is themechanism obvious but it does suggest that the wellbeing ofthe muscle fiber may be influenced by the mechanismsassociated with myogenic growth. Perhaps, the reducedproportion of MPC available for fiber repair in ReX musclespredisposes them to excessive damage. Accordingly, newlyfused MPCs could inhibit damaged fiber destruction eventhough these MPC do not express dystrophin, for example bytheir elevated level of utrophin, which has been shown to becapable of substituting functionally for dystrophin. A pro-gressive exhaustion of the MPC pool may also explain whythe muscle weight differences between mdx-c and ReXappeared only after the age of two months. Prior to theonset of necrosis, myogenic cells would be needed only forgrowth, but thereafter, during the acute disease phase, thereduced availability of MPC in the ReX mouse might become acritical factor, eventually resulting in a diminished regenera-tion and repair of fibers, a greater fibers degeneration and aloss of muscle mass.

Mdr1-dependent mSP activity in dystrophic muscles

To gain an insight into the molecular mechanism thatmediates the mdr1 dependent muscle regeneration activity,we have thoroughly analyzed the bonemarrow andmuscle SPcontent of four different mice strains. In agreement withprevious studies our results confirmed that in themdr1ab(−/−)mouse the proportion of SP cells is not less than normal ineither bone marrow or the muscles, thus supporting the viewthat the SP phenotype in these tissues is independent of mdr1expression. In regenerating muscle, however, the situation isclearly different than in normal muscle, because in the ReXmice we have identified a highly reproducible reduction in theproportion of mSP. Indeed, cellular exclusion of Hoechst 3342by mdr1 is not without precedent. First, mdr1 overexpressionhas been shown to produce the SP phenotype in mouse bonemarrow [36] and myoblast cultures [30]. Second, in myoblastcultures, an expansion of the SP population by FGF6

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overexpression has been shown to be mdr1-dependent [8].Third, the SP phenotype in cells derived frommammary glandand bone marrow has been shown to be dependent on bothmdr1 and ABCG2 expression [24,37]. Fourth, the recentsuggestion [47], that in muscle the ABCG2 transporter is notthe only molecule accounting for the mSP phenotype, issupported by the demonstration of Tadjali et al. [48], that inmuscle SP only 35% of the non-hematopoietic cells expressedABCG2. We propose that the composition of the Hoechst-excluding (SP) cell population derived from dystrophic muscleis profoundly distinct from the mSP cell population derivedfrom normal muscle, which could be explained by therelatively fast MPC turnover in dystrophic muscle comparedto that of normal adult mouse muscle. This proposal isstrongly supported by the superior myogenic potential ofmSP cells derived from dystrophic muscle (see below).Furthermore, the unchanged level of ABCG2 expressionbetween ReX and mdx-c muscles suggested that the lowermSP proportion in ReXmuscle results directly from the loss ofmdr1-dependent Hoechst exclusion activity. The distributionof the cells within the mSP gate in the ReX muscle suggestedthat in dystrophic muscles the ABCG2 may be the principaltransporter responsible for the distal mSP, but that the mdr1transporter accounts for the proximal mSP activity. Interest-ingly, the same seems to be true also in the mammary glandmSP population (see Fig. 1 in [37]). In conclusion, we proposethatwhile the Abcg2/Bcrp1 gene product is themajormoleculeresponsible for the SP phenotype in normal muscles, indystrophic muscle the SP phenotype is also strongly depen-dent on mdr1 expression.

Myogenic potential of mSP

An early study performed with in a non-dystrophic mousereported that mSP cells could not differentiate in vitro intomultinucleated myotubes unless they were co-cultured withprimary myoblasts [49]. More recently, Meeson et al. [40]demonstrated that mSP cells from non-dystrophic mousecould differentiate in vitro into multinucleated musclemyotubes, when cultured on matrigel. In the presentstudy, C57BL/6 mSP cultured on matrigel produced only anoccasional multinucleated myotube, confirming the resultsof Meeson et al. Surprisingly, SP cells derived fromdystrophic muscles differentiated efficiently into a myotubenetwork. Thus mSP derived from dystrophic muscles areprofoundly distinct from normal muscle-derived SP cells andcould be an important component in the efficient regenera-tion mechanism of mdx muscle. These results suggest thatthe lower mSP content in ReX compared to mdx-c micecould contribute to the reduced muscle regeneration capa-city in ReX.

The clear identity of the mdr1 positive MPC which isdeficient in the muscles of the ReX mice is still unknown. Ourimmunostaining results suggest that the mdr1 expressingcells are distinct from satellite cells; however, we cannotexclude a lower level of mdr1 expression by satellite cells. Acommon view is that the large majority of the muscleregenerative potential resides within the satellite cell popula-tion, though some muscle regeneration activity could origi-nate outside of the satellite cell compartment (reviewed in

[50]). The participation of interstitial mSP in muscle regenera-tion has recently been shown in other studies [40,47];however, the mSP cells in these studies were derived fromnon-regeneratingmuscle and their interstitial localization hasbased on ABCG2 staining. Our data are compatible with theidea thatmdr1 +vemuscle interstitial cells participate activelyin regeneration of dystrophic muscle. Nevertheless at themoment we cannot exclude the possibility that mdr1expressed also by satellite cells, or a subpopulation of thesatellite cells, and that the absence of mdr1 expressioninhibits muscle regeneration via its effect on satellite cells.Indeed activation of quiescent muscle satellite cell requiressphingolipid signaling [51], while as the mdr1 is a lipidtranslocase of a broad specificity [52]. Further study will benecessary to clarify this point.

Reduced content of MPC in ReX skeletal muscles

We have shown that dystrophic muscle-derived SP cells arehighlymyogenic. Yet, mSPs represent only 1–2% of themuscleMMNC and therefore do not constitute the major cellularreservoir available for muscle regeneration. In order tocharacterize further the muscle regeneration differencesbetween ReX and mdx-c one must analyze the myogenicpotential residing within the entire non-fractionated MMNC.Striated muscle is a complex tissue and only a fraction of theMMNC are muscle MPCs. To analyze directly and quantita-tively the content of MPC in the muscles of ReX compared tomdx-c, we have used two different myogenic markers. Thefirst one, the muscle-specific intermediate filament proteindesmin is not only a myofiber structural protein but also oneof the earliest markers of activated satellite cells [41,42]. Thesecond marker is the SM/C-2.6 antibody recently developedand characterized against mouse muscle satellite cells [31].Having initially established that in the mdx-c and ReX(dystrophic muscles), the SM/C-2.6 “bright”, CD45 −ve stainedcells represent themajor myogenic potential, we then showedthat the proportion of both the desmin-positive and the SM/C-2.6 “bright”, CD45 −ve cells was drastically lower in ReX thanin mdx-c. Therefore, in ReX muscles, not only have we foundhalf the normal mSP content but also the ReX's non-fractionated MMNC displayed reduced myogenic potentialcompared to mdx-c. The increased proportion of CD45 +vecells identified in the ReX MMNC could perhaps result fromthe higher inflammatory state of these muscles. Therefore indystrophic muscle, mdr1 expression is required for thepreservation of both mSP and myogenic MP cells. We havepreviously shown that in cultivated myoblasts the mSP cellscan give rise to mMP cells that could eventually differentiateinto myotubes [30]. Other groups have shown that mSPderived from normal muscles can integrate and contribute toregenerated muscle fibers in vivo [35,40,49,53]. Lastly, in thepresent study, we have provided evidence for the superiormyogenic activity of the mSP cells derived from dystrophicmuscle over those derived from normal muscle. Takentogether, it is likely that in the mdx-c muscle some of themSP cells are highly myogenic and that in the ReX muscle thereduced mSP proportion is a major reason for the reducedproportion of the myogenic MMNC. The exact relation indystrophic muscle between mSP and satellite cells and

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between mSP and the myogenic MP cells remains to beestablished.

A biological role for mdr1 in a stress-induced regenerationprogram?

Mdr1 expression is not vital under normal conditions, asindicated by themild symptoms of themdr1-null mice [10,24],but becomes important under the conditions used in thisstudy, in which regeneration of chronically damaged muscleswas dependent on a constant supply of stem cells. Theseobservations support a model in which mdr1 expression instem cells is dispensable for tissue homeostasis in normalsituations but becomes necessary under extreme stressconditions where it is induced by regeneration factors as forexample FGF6 in regenerating muscles [8]. Accordingly, thisview mdr1 would play a key role in the tissue regenerationprogram by the preservation of stem cells. Involvement ofcancer stem cells, some of which have an SP phenotype [54] inthe pathology of cancer, suggests a similar role for mdr1 in theevolution of cancer pathologies [55].

Acknowledgments

The authors are grateful to Dr Terence Partridge for his helpwith the DD manipulation, the anti-dystrophin antisera, andfor critical reading of the manuscript. We thank XavierBroudeur, Laetitia Van Wittenberghe, Julien Picot, ChristopheGeorger, Daniel Stockholm, and Corinne Laplace-Builhé fortheir excellent technical help. The work was supported bygrants from the AFM and the CNRS.

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at doi:10.1016/j.yexcr.2007.02.036.

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